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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Structural studies of neuropilin‐2 reveal a zinc ion binding site remote from the vascular endothelial growth factor binding pocket</title><author><name sortKey="Tsai, Yi Hun Isabella" sort="Tsai, Yi Hun Isabella" uniqKey="Tsai Y" first="Yi-Chun Isabella" last="Tsai">Yi-Chun Isabella Tsai</name><affiliation><nlm:aff id="febs13711-aff-0001"></nlm:aff></affiliation><affiliation><nlm:aff id="febs13711-curr-0001"></nlm:aff></affiliation></author><author><name sortKey="Fotinou, Constantina" sort="Fotinou, Constantina" uniqKey="Fotinou C" first="Constantina" last="Fotinou">Constantina Fotinou</name><affiliation><nlm:aff id="febs13711-aff-0002"></nlm:aff></affiliation></author><author><name sortKey="Rana, Rohini" sort="Rana, Rohini" uniqKey="Rana R" first="Rohini" last="Rana">Rohini Rana</name><affiliation><nlm:aff id="febs13711-aff-0002"></nlm:aff></affiliation></author><author><name sortKey="Yelland, Tamas" sort="Yelland, Tamas" uniqKey="Yelland T" first="Tamas" last="Yelland">Tamas Yelland</name><affiliation><nlm:aff id="febs13711-aff-0001"></nlm:aff></affiliation></author><author><name sortKey="Frankel, Paul" sort="Frankel, Paul" uniqKey="Frankel P" first="Paul" last="Frankel">Paul Frankel</name><affiliation><nlm:aff id="febs13711-aff-0003"></nlm:aff></affiliation></author><author><name sortKey="Zachary, Ian" sort="Zachary, Ian" uniqKey="Zachary I" first="Ian" last="Zachary">Ian Zachary</name><affiliation><nlm:aff id="febs13711-aff-0003"></nlm:aff></affiliation></author><author><name sortKey="Djordjevic, Snezana" sort="Djordjevic, Snezana" uniqKey="Djordjevic S" first="Snezana" last="Djordjevic">Snezana Djordjevic</name><affiliation><nlm:aff id="febs13711-aff-0001"></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">26991001</idno><idno type="pmc">4914954</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914954</idno><idno type="RBID">PMC:4914954</idno><idno type="doi">10.1111/febs.13711</idno><date when="2016">2016</date><idno type="wicri:Area/Pmc/Corpus">000739</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000739</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Structural studies of neuropilin‐2 reveal a zinc ion binding site remote from the vascular endothelial growth factor binding pocket</title><author><name sortKey="Tsai, Yi Hun Isabella" sort="Tsai, Yi Hun Isabella" uniqKey="Tsai Y" first="Yi-Chun Isabella" last="Tsai">Yi-Chun Isabella Tsai</name><affiliation><nlm:aff id="febs13711-aff-0001"></nlm:aff></affiliation><affiliation><nlm:aff id="febs13711-curr-0001"></nlm:aff></affiliation></author><author><name sortKey="Fotinou, Constantina" sort="Fotinou, Constantina" uniqKey="Fotinou C" first="Constantina" last="Fotinou">Constantina Fotinou</name><affiliation><nlm:aff id="febs13711-aff-0002"></nlm:aff></affiliation></author><author><name sortKey="Rana, Rohini" sort="Rana, Rohini" uniqKey="Rana R" first="Rohini" last="Rana">Rohini Rana</name><affiliation><nlm:aff id="febs13711-aff-0002"></nlm:aff></affiliation></author><author><name sortKey="Yelland, Tamas" sort="Yelland, Tamas" uniqKey="Yelland T" first="Tamas" last="Yelland">Tamas Yelland</name><affiliation><nlm:aff id="febs13711-aff-0001"></nlm:aff></affiliation></author><author><name sortKey="Frankel, Paul" sort="Frankel, Paul" uniqKey="Frankel P" first="Paul" last="Frankel">Paul Frankel</name><affiliation><nlm:aff id="febs13711-aff-0003"></nlm:aff></affiliation></author><author><name sortKey="Zachary, Ian" sort="Zachary, Ian" uniqKey="Zachary I" first="Ian" last="Zachary">Ian Zachary</name><affiliation><nlm:aff id="febs13711-aff-0003"></nlm:aff></affiliation></author><author><name sortKey="Djordjevic, Snezana" sort="Djordjevic, Snezana" uniqKey="Djordjevic S" first="Snezana" last="Djordjevic">Snezana Djordjevic</name><affiliation><nlm:aff id="febs13711-aff-0001"></nlm:aff></affiliation></author></analytic><series><title level="j">The Febs Journal</title><idno type="ISSN">1742-464X</idno><idno type="eISSN">1742-4658</idno><imprint><date when="2016">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Neuropilin‐2 is a transmembrane receptor involved in lymphangiogenesis and neuronal development. In adults, neuropilin‐2 and its homologous protein neuropilin‐1 have been implicated in cancers and infection. Molecular determinants of the ligand selectivity of neuropilins are poorly understood. We have identified and structurally characterized a zinc ion binding site on human neuropilin‐2. The neuropilin‐2‐specific zinc ion binding site is located near the interface between domains b1 and b2 in the ectopic region of the protein, remote from the neuropilin binding site for its physiological ligand, i.e. vascular endothelial growth factor. We also present an X‐ray crystal structure of the neuropilin‐2 b1 domain in a complex with the C‐terminal sub‐domain of <styled-content style="fixed-case">VEGF</styled-content>‐A. Zn<sup>2+</sup> binding to neuropilin‐2 destabilizes the protein structure but this effect was counteracted by heparin, suggesting that modifications by glycans and zinc in the extracellular matrix may affect functional neuropilin‐2 ligand binding and signalling activity.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Read, R" uniqKey="Read R">R Read</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">FEBS J</journal-id><journal-id journal-id-type="iso-abbrev">FEBS J</journal-id><journal-id journal-id-type="doi">10.1111/(ISSN)1742-4658</journal-id><journal-id journal-id-type="publisher-id">FEBS</journal-id><journal-title-group><journal-title>The Febs Journal</journal-title></journal-title-group><issn pub-type="ppub">1742-464X</issn><issn pub-type="epub">1742-4658</issn><publisher><publisher-name>John Wiley and Sons Inc.</publisher-name><publisher-loc>Hoboken</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">26991001</article-id><article-id pub-id-type="pmc">4914954</article-id><article-id pub-id-type="doi">10.1111/febs.13711</article-id><article-id pub-id-type="publisher-id">FEBS13711</article-id><article-categories><subj-group subj-group-type="overline"><subject>Original Article</subject></subj-group><subj-group subj-group-type="heading"><subject>Original Articles</subject></subj-group></article-categories><title-group><article-title>Structural studies of neuropilin‐2 reveal a zinc ion binding site remote from the vascular endothelial growth factor binding pocket</article-title><alt-title alt-title-type="left-running-head">Y.‐C. I. Tsai <italic>et al</italic>.</alt-title></title-group><contrib-group><contrib id="febs13711-cr-0001" contrib-type="author"><name><surname>Tsai</surname><given-names>Yi‐Chun Isabella</given-names></name><xref ref-type="aff" rid="febs13711-aff-0001"><sup>1</sup></xref><xref ref-type="aff" rid="febs13711-curr-0001"><sup>4</sup></xref></contrib><contrib id="febs13711-cr-0002" contrib-type="author"><name><surname>Fotinou</surname><given-names>Constantina</given-names></name><xref ref-type="aff" rid="febs13711-aff-0002"><sup>2</sup></xref></contrib><contrib id="febs13711-cr-0003" contrib-type="author"><name><surname>Rana</surname><given-names>Rohini</given-names></name><xref ref-type="aff" rid="febs13711-aff-0002"><sup>2</sup></xref></contrib><contrib id="febs13711-cr-0004" contrib-type="author"><name><surname>Yelland</surname><given-names>Tamas</given-names></name><xref ref-type="aff" rid="febs13711-aff-0001"><sup>1</sup></xref></contrib><contrib id="febs13711-cr-0005" contrib-type="author"><name><surname>Frankel</surname><given-names>Paul</given-names></name><xref ref-type="aff" rid="febs13711-aff-0003"><sup>3</sup></xref></contrib><contrib id="febs13711-cr-0006" contrib-type="author"><name><surname>Zachary</surname><given-names>Ian</given-names></name><xref ref-type="aff" rid="febs13711-aff-0003"><sup>3</sup></xref></contrib><contrib id="febs13711-cr-0007" contrib-type="author" corresp="yes"><name><surname>Djordjevic</surname><given-names>Snezana</given-names></name><xref ref-type="aff" rid="febs13711-aff-0001"><sup>1</sup></xref></contrib></contrib-group><aff id="febs13711-aff-0001"><label><sup>1</sup></label><named-content content-type="organisation-division">Institute of Structural and Molecular Biology</named-content><institution>University College London</institution><named-content content-type="city">London</named-content><country country="GB">UK</country></aff><aff id="febs13711-aff-0002"><label><sup>2</sup></label><named-content content-type="organisation-division">Magnus Life Science</named-content><institution>University College London</institution><named-content content-type="city">London</named-content><country country="GB">UK</country></aff><aff id="febs13711-aff-0003"><label><sup>3</sup></label><named-content content-type="organisation-division">Centre for Cardiovascular Biology & Medicine</named-content><institution>BHF Laboratories at University College London</institution><named-content content-type="city">London</named-content><country country="GB">UK</country></aff><aff id="febs13711-curr-0001"><label><sup>4</sup></label>Present address:<named-content content-type="organisation-division">Francis Crick Institute</named-content><institution>Mill Hill Laboratory</institution><named-content content-type="city">The Ridgeway</named-content><named-content content-type="city">London</named-content><named-content content-type="post-code">NW7 1AA</named-content><country country="GB">UK</country></aff><author-notes><corresp id="correspondenceTo"><label>*</label><bold>Correspondence</bold><break></break>S. Djordjevic, Institute of Structural and Molecular Biology, University College London, Darwin Building, Gower Street, London WC1E 6BT, UK<break></break>Fax: +44 20 7679 7193<break></break>Tel: +44 20 7679 2230<break></break>E‐mail: <email>s.djordjevic@ucl.ac.uk</email><break></break></corresp></author-notes><pub-date pub-type="epub"><day>01</day><month>4</month><year>2016</year></pub-date><pub-date pub-type="ppub"><month>5</month><year>2016</year></pub-date><volume>283</volume><issue>10</issue><issue-id pub-id-type="doi">10.1111/febs.2016.283.issue-10</issue-id><fpage>1921</fpage><lpage>1934</lpage><history><date date-type="received"><day>24</day><month>12</month><year>2015</year></date><date date-type="rev-recd"><day>24</day><month>2</month><year>2016</year></date><date date-type="accepted"><day>10</day><month>3</month><year>2016</year></date></history><permissions><pmc-comment> Copyright © 2016 Federation of European Biochemical Societies </pmc-comment>
<copyright-statement content-type="article-copyright">© 2016 The Authors. The <italic>FEBS</italic> Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.</copyright-statement><license license-type="creativeCommonsBy"><license-p>This is an open access article under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution</ext-link> License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri content-type="pdf" xlink:type="simple" xlink:href="file:FEBS-283-1921.pdf"></self-uri><abstract id="febs13711-abs-0001"><p>Neuropilin‐2 is a transmembrane receptor involved in lymphangiogenesis and neuronal development. In adults, neuropilin‐2 and its homologous protein neuropilin‐1 have been implicated in cancers and infection. Molecular determinants of the ligand selectivity of neuropilins are poorly understood. We have identified and structurally characterized a zinc ion binding site on human neuropilin‐2. The neuropilin‐2‐specific zinc ion binding site is located near the interface between domains b1 and b2 in the ectopic region of the protein, remote from the neuropilin binding site for its physiological ligand, i.e. vascular endothelial growth factor. We also present an X‐ray crystal structure of the neuropilin‐2 b1 domain in a complex with the C‐terminal sub‐domain of <styled-content style="fixed-case">VEGF</styled-content>‐A. Zn<sup>2+</sup> binding to neuropilin‐2 destabilizes the protein structure but this effect was counteracted by heparin, suggesting that modifications by glycans and zinc in the extracellular matrix may affect functional neuropilin‐2 ligand binding and signalling activity.</p></abstract><kwd-group kwd-group-type="author-generated"><kwd id="febs13711-kwd-0001">neuropilins</kwd><kwd id="febs13711-kwd-0002">signalling</kwd><kwd id="febs13711-kwd-0003">vascular endothelial growth factor</kwd><kwd id="febs13711-kwd-0004">X‐ray crystallography</kwd><kwd id="febs13711-kwd-0005">zinc</kwd></kwd-group><funding-group><award-group><funding-source>British Heart Foundation</funding-source><award-id>PG/10/52/28448</award-id></award-group></funding-group><funding-group><award-group><funding-source>Magnus Life Science</funding-source></award-group></funding-group><counts><page-count count="14"></page-count></counts><custom-meta-group><custom-meta><meta-name>source-schema-version-number</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta><meta-name>component-id</meta-name><meta-value>febs13711</meta-value></custom-meta><custom-meta><meta-name>cover-date</meta-name><meta-value>May 2016</meta-value></custom-meta><custom-meta><meta-name>details-of-publishers-convertor</meta-name><meta-value>Converter:WILEY_ML3GV2_TO_NLMPMC version:4.9.1 mode:remove_FC converted:23.06.2016</meta-value></custom-meta></custom-meta-group></article-meta></front><body><def-list list-content="abbreviations" id="febs13711-dl-0001"><title>Abbreviations</title><def-item><term>HBD</term><def><p>heparin binding domain</p></def></def-item><def-item><term>NRP1</term><def><p>neuropilin‐1</p></def></def-item><def-item><term>NRP2</term><def><p>neuropilin‐2</p></def></def-item><def-item><term>VEGF</term><def><p>vascular endothelial growth factor</p></def></def-item></def-list><sec id="febs13711-sec-0001"><title>Introduction</title><p>Neuropilin‐2 (NRP2) and neuropilin‐1 (NRP1) are homologous transmembrane receptors that are expressed in diverse tissues, sharing a common domain organization and 44% amino acid sequence identity in humans <xref rid="febs13711-bib-0001" ref-type="ref">1</xref>, <xref rid="febs13711-bib-0002" ref-type="ref">2</xref>, <xref rid="febs13711-bib-0003" ref-type="ref">3</xref>. Both NRPs have essential roles in the control of axonal homing in the developing nervous system, as revealed by the phenotypes of genetically deficient mice <xref rid="febs13711-bib-0004" ref-type="ref">4</xref>, <xref rid="febs13711-bib-0005" ref-type="ref">5</xref>, <xref rid="febs13711-bib-0006" ref-type="ref">6</xref>, <xref rid="febs13711-bib-0007" ref-type="ref">7</xref>. NRP1 is also required for embryonic cardiovascular development, whereas NRP2 has a more restricted developmental role in lymphangiogenesis <xref rid="febs13711-bib-0007" ref-type="ref">7</xref>. In addition, neuropilins have been implicated in other physiological and disease‐related processes in adult organisms, such as various cancers, proliferative retinopathies and immunomodulation <xref rid="febs13711-bib-0008" ref-type="ref">8</xref>.</p><p>Neuropilins mediate signalling by binding to vascular endothelial growth factors (VEGFs) and semaphorins, secreted ligands that are essential for cardiovascular and neuronal development, respectively <xref rid="febs13711-bib-0009" ref-type="ref">9</xref>, <xref rid="febs13711-bib-0010" ref-type="ref">10</xref>, <xref rid="febs13711-bib-0011" ref-type="ref">11</xref>, <xref rid="febs13711-bib-0012" ref-type="ref">12</xref>, <xref rid="febs13711-bib-0013" ref-type="ref">13</xref>, <xref rid="febs13711-bib-0014" ref-type="ref">14</xref>. The ligand specificities for NRPs are often found overlap in cell‐based studies, whereas NRP1 and NRP2 display distinct expression patterns and ligand preferences <italic>in vivo</italic><xref rid="febs13711-bib-0006" ref-type="ref">6</xref>, <xref rid="febs13711-bib-0011" ref-type="ref">11</xref>, <xref rid="febs13711-bib-0015" ref-type="ref">15</xref>, <xref rid="febs13711-bib-0016" ref-type="ref">16</xref>. In the central nervous system, for example, the semaphorin Sema3A preferentially signals via NRP1 to control cortical neuron basal dendritic arborization, whereas Sema3F binds NRP2 to regulate formation of apical dendrite processes of cortical neurons <xref rid="febs13711-bib-0017" ref-type="ref">17</xref>. In the vascular system, the ligand preferences for NRPs are less clearly defined, but the VEGF‐A splice variant VEGF‐A<sub>165</sub> appears to be a major ligand for NRP1 on endothelial cells, whereas VEGF‐C signals via NRP2 in lymphatic vessels <xref rid="febs13711-bib-0009" ref-type="ref">9</xref>, <xref rid="febs13711-bib-0011" ref-type="ref">11</xref>, <xref rid="febs13711-bib-0018" ref-type="ref">18</xref>, <xref rid="febs13711-bib-0019" ref-type="ref">19</xref>, <xref rid="febs13711-bib-0020" ref-type="ref">20</xref>. Furthermore, NRP2 is co‐expressed with NRP1 in endothelial and other cell types, NRP2 and NRP1 may associate to form heterodimers <xref rid="febs13711-bib-0014" ref-type="ref">14</xref>, and NRP2 also binds to VEGF‐A in trimeric complexes with VEGF receptor 2 <xref rid="febs13711-bib-0021" ref-type="ref">21</xref>, <xref rid="febs13711-bib-0022" ref-type="ref">22</xref>, suggesting that NRPs have the potential to interact in a cooperative fashion in the endothelium.</p><p>The protein regions of VEGF‐A encoded by exons 2‐5 form a cysteine knot structure that interacts with VEGF receptors, while the 55 amino acid residue C‐terminal domain of VEGF‐A, encoded by exons 7 and 8, is required for binding to neuropilin and heparin <xref rid="febs13711-bib-0023" ref-type="ref">23</xref>, <xref rid="febs13711-bib-0024" ref-type="ref">24</xref>. Several structural and biochemical studies have addressed the molecular basis of the VEGF binding selectivity of neuropilins. In all of the available structures, including those of a small molecule peptidomimetic in complex with NRP1 and of a fusion of the NRP1 binding region of VEGF‐A with the NRP1 b1 domain, a key feature is binding of a C‐terminal arginine residue to a conserved region of the b1 domain of neuropilin <xref rid="febs13711-bib-0025" ref-type="ref">25</xref>, <xref rid="febs13711-bib-0026" ref-type="ref">26</xref>, <xref rid="febs13711-bib-0027" ref-type="ref">27</xref>. Crystal structures of a fusion of human NRP1 b1 (NRP1 residues 274–429) with the heparin binding domain (HBD) of VEGF‐A<sub>165</sub> (VEGF‐A<sub>165</sub>‐HBD; VEGF‐A<sub>165</sub> residues 115–165) <xref rid="febs13711-bib-0026" ref-type="ref">26</xref> or of human NRP2 domains b1b2 (NRP2 residues 276–595) fused with the C‐terminal five amino acids of mature VEGF‐C (SIIRR, residues 219–223) <xref rid="febs13711-bib-0027" ref-type="ref">27</xref> revealed a high degree of similarity with respect to the interactions of the C‐terminal arginine residue of VEGF‐A<sub>165</sub> and VEGF‐C with NRP1 and NRP2, respectively. Initial site‐directed mutagenesis experiments addressing sequence differences in the binding sites of NRP1 and NRP2 have led to the proposal that a charge repulsion mechanism provides the molecular basis for ligand selectivity <xref rid="febs13711-bib-0026" ref-type="ref">26</xref>, <xref rid="febs13711-bib-0027" ref-type="ref">27</xref>. However, more comprehensive structural and functional studies of NRP2, including other members of the VEGF ligand family, are needed to fully characterize and understand the molecular basis for both VEGF binding to NRP2 and differences between NRP1 and NRP2 in terms of ligand preference and biological function.</p><p>We now report structural and biochemical characterization of a conserved Zn<sup>2+</sup> ion binding site on NRP2. This binding site is not present in NRP1, and we hypothesize that it may be involved in modulation of ligand binding to NRP2. In addition, we describe the three‐dimensional structure of a molecular complex between the C‐terminal region of VEGF‐A<sub>165</sub> and the b1 ligand binding domain of NRP2, further addressing the question of ligand selectivity.</p></sec><sec id="febs13711-sec-0002"><title>Results</title><sec id="febs13711-sec-0003"><title>Crystal structure reveals a NRP2‐specific zinc ion binding site</title><p>We have solved a structure of NRP2 from the crystal grown in the presence of zinc sulfate that diffracted to 1.8 Å resolution (Fig. <xref rid="febs13711-fig-0001" ref-type="fig">1</xref> and Table <xref rid="febs13711-tbl-0001" ref-type="table-wrap">1</xref>). No specific peptidic ligand was present during the crystallization, but the ligand binding pocket showed a difference electron density that was consistent with a molecule of MES buffer (2‐ethanesulfonic acid) present in the mother liquor, although with the significant disorder of the morpholine ring (Fig. <xref rid="febs13711-fig-0001" ref-type="fig">1</xref>). In addition, three zinc ion binding sites were identified in the difference electron density maps, and the location of zinc ions was verified by an anomalous difference map (Fig. <xref rid="febs13711-fig-0001" ref-type="fig">1</xref>A). The most significant zinc ion binding site (referred to as site ‘1’) is formed by the side chains of the amino acids Tyr375, His377 and His399, with the fourth zinc ion coordination most likely provided by a chloride ion from the protein buffer solution used for crystallization (Fig. <xref rid="febs13711-fig-0002" ref-type="fig">2</xref>A). We interpreted the density as representing a chloride ion, as when water molecule was modelled in that position, there was always strong residual electron density left. The other two zinc ions bound to NRP2 in this crystal structure are less likely to be biologically relevant, and their binding sites have probably been generated by the crystallization conditions and crystal packing. Both of the additional two binding sites are formed within the inter‐molecular space, with two zinc‐coordinating side chains provided by one molecule and the third side chain provided by an amino acid of the crystallographic neighbour. Specifically, the second zinc ion is coordinated by Glu284 and His312 from one molecule and His415 from the neighbouring molecule; the third zinc ion is coordinated by Lys362 and His381 from one molecule and Glu370 from the neighbouring molecule. The zinc‐coordinating residues at site 1 of NRP2 are conserved throughout mammalian species and unique to NRP2 as they are absent in NRP1 sequences (Fig. <xref rid="febs13711-fig-0002" ref-type="fig">2</xref>B), further suggesting a specific physiological role for this metal binding site. The orientation of the three coordinating side chains in the presence of zinc ion within our structure is different from that of the side chains of equivalent residues in the previously reported NRP2 structure <xref rid="febs13711-bib-0028" ref-type="ref">28</xref>, reflecting adjustment of the side chain conformations in order to accommodate ion binding.</p><fig fig-type="Figure" xml:lang="en" id="febs13711-fig-0001" orientation="portrait" position="float"><label>Figure 1</label><caption><p>Zn<sup>2+</sup> binding site on <styled-content style="fixed-case">NRP</styled-content>2. (A) The X‐ray crystal structure of <styled-content style="fixed-case">NRP</styled-content>2 crystallized in the presence of zinc revealed three zinc ions bound to the b1 domain. The structure is shown in yellow ribbon representation, and the three zinc ions are shown as grey spheres. A molecule of <styled-content style="fixed-case">MES</styled-content> (2‐ethanesulfonic acid) bound to the canonical ligand binding site on the neuropilin b1 domain is shown using sticks representation. Loops L1 and L3 that shape the binding cleft are indicated. (B) Enlarged view of the ligand binding site, with the <styled-content style="fixed-case">MES</styled-content>‐associated 2 <italic>F</italic><sub>o</sub> – <italic>F</italic><sub>c</sub> electron density map shown as a grey wire mesh and contoured at 1 σ.</p></caption><graphic id="nlm-graphic-3" xlink:href="FEBS-283-1921-g001"></graphic></fig><table-wrap id="febs13711-tbl-0001" xml:lang="en" orientation="portrait" position="float"><label>Table 1</label><caption><p>Data collection and refinement statistics. Values in parentheses are for the highest‐resolution shell</p></caption><table frame="hsides" rules="groups"><col style="border-right:solid 1px #000000" span="1"></col><col style="border-right:solid 1px #000000" span="1"></col><col style="border-right:solid 1px #000000" span="1"></col><thead valign="top"><tr style="border-bottom:solid 1px #000000"><th align="left" valign="top" rowspan="1" colspan="1">Crystal</th><th align="left" valign="top" rowspan="1" colspan="1">EG00086‐bound</th><th align="left" valign="top" rowspan="1" colspan="1">Zn<sup>2+</sup>‐bound</th></tr></thead><tbody><tr><td align="left" colspan="3" rowspan="1">Data collection</td></tr><tr><td align="left" style="padding-left:10%" rowspan="1" colspan="1">X‐ray source</td><td align="left" rowspan="1" colspan="1">Diamond I03</td><td align="left" rowspan="1" colspan="1">ESRF ID23‐2</td></tr><tr><td align="left" style="padding-left:10%" rowspan="1" colspan="1">Space group</td><td align="left" rowspan="1" colspan="1">C222<sub>1</sub></td><td align="left" rowspan="1" colspan="1">C222<sub>1</sub></td></tr><tr><td align="left" colspan="3" rowspan="1">Cell constants</td></tr><tr><td align="left" style="padding-left:10%" rowspan="1" colspan="1"><italic>a</italic>,<italic> b</italic>,<italic> c</italic> (Å)</td><td align="left" rowspan="1" colspan="1">110.25, 139.52, 106.04</td><td align="left" rowspan="1" colspan="1">99.39, 105.27, 45.75</td></tr><tr><td align="left" style="padding-left:10%" rowspan="1" colspan="1">α, β, γ (°)</td><td align="left" rowspan="1" colspan="1">90.00, 90.00, 90.00</td><td align="left" rowspan="1" colspan="1">90.00, 90.00, 90.00</td></tr><tr><td align="left" rowspan="1" colspan="1">Resolution (Å)</td><td align="left" rowspan="1" colspan="1">67.03–1.95 (2.00–1.95)</td><td align="left" rowspan="1" colspan="1">36.13–1.80 (1.84–1.80)</td></tr><tr><td align="left" rowspan="1" colspan="1">Completeness (%)</td><td align="left" rowspan="1" colspan="1">99.5 (97.4)</td><td align="left" rowspan="1" colspan="1">99.9 (99.8)</td></tr><tr><td align="left" rowspan="1" colspan="1">Multiplicity</td><td align="left" rowspan="1" colspan="1">3.4 (3.0)</td><td align="left" rowspan="1" colspan="1">4.6 (4.5)</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>R</italic><sub>merge</sub></td><td align="left" rowspan="1" colspan="1">0.059 (0.223)</td><td align="left" rowspan="1" colspan="1">0.052 (0.230)</td></tr><tr><td align="left" rowspan="1" colspan="1"><<italic>I</italic>/σ(<italic>I</italic>)></td><td align="left" rowspan="1" colspan="1">10.8 (4.3)</td><td align="left" rowspan="1" colspan="1">16.5 (5.4)</td></tr><tr><td align="left" rowspan="1" colspan="1">CC<sub>½</sub></td><td align="left" rowspan="1" colspan="1">0.996 (0.944)</td><td align="left" rowspan="1" colspan="1">0.997 (0.937)</td></tr><tr><td align="left" rowspan="1" colspan="1">Wilson <italic>B</italic> factor (Å<sup>2</sup>)</td><td align="left" rowspan="1" colspan="1">23.3</td><td align="left" rowspan="1" colspan="1">21.7</td></tr><tr><td align="left" colspan="3" rowspan="1">Refinement</td></tr><tr><td align="left" style="padding-left:10%" rowspan="1" colspan="1">Number of reflections</td><td align="left" rowspan="1" colspan="1">59 661</td><td align="left" rowspan="1" colspan="1">22 736</td></tr><tr><td align="left" style="padding-left:10%" rowspan="1" colspan="1"><italic>R</italic><sub>work</sub>/<italic>R</italic><sub>free</sub></td><td align="left" rowspan="1" colspan="1">0.204/0.234</td><td align="left" rowspan="1" colspan="1">0.177/0.217</td></tr><tr><td align="left" style="padding-left:10%" rowspan="1" colspan="1">Mean <italic>B</italic> value, all atoms (Å<sup>2</sup>)</td><td align="left" rowspan="1" colspan="1">26.0</td><td align="left" rowspan="1" colspan="1">25.5</td></tr><tr><td align="left" colspan="3" rowspan="1">RMSDs</td></tr><tr><td align="left" style="padding-left:10%" rowspan="1" colspan="1">Bond lengths (Å)</td><td align="left" rowspan="1" colspan="1">0.010</td><td align="left" rowspan="1" colspan="1">0.016</td></tr><tr><td align="left" style="padding-left:10%" rowspan="1" colspan="1">Bond angles (°)</td><td align="left" rowspan="1" colspan="1">1.463</td><td align="left" rowspan="1" colspan="1">1.753</td></tr><tr><td align="left" colspan="3" rowspan="1">Ramachandran plot (%)</td></tr><tr><td align="left" style="padding-left:10%" rowspan="1" colspan="1">Favoured, allowed,</td><td align="left" rowspan="1" colspan="1">96, 4, 0 (chain A)</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" style="padding-left:10%" rowspan="1" colspan="1">outliers</td><td align="left" rowspan="1" colspan="1">95, 5, 0 (chain B)</td><td align="left" rowspan="1" colspan="1">96, 4, 0</td></tr></tbody></table><permissions><copyright-holder>John Wiley & Sons, Ltd</copyright-holder></permissions></table-wrap><fig fig-type="Figure" xml:lang="en" id="febs13711-fig-0002" orientation="portrait" position="float"><label>Figure 2</label><caption><p>The main zinc ion binding site is <styled-content style="fixed-case">NRP</styled-content>2‐specific. (A) Stereoimage of the <styled-content style="fixed-case">NRP</styled-content>2 residues involved in Zn<sup>2+</sup> ion coordination: Y375, H377 and H399. The residues are shown using stick representation within the final 2 <italic>F</italic><sub>o</sub> – <italic>F</italic><sub>c</sub> electron density map contoured at 1 σ (grey mesh). (B) Sequence alignment for the part of the neuropilin sequences that includes all three residues that bind zinc in site ‘1’ shows that the site is conserved in <styled-content style="fixed-case">NRP</styled-content>2 and absent in <styled-content style="fixed-case">NRP</styled-content>1. The residues associated with <styled-content style="fixed-case">NRP</styled-content>2 are highlighted in yellow, and the residues in the analogous positions in <styled-content style="fixed-case">NRP</styled-content>1 are highlighted in green.</p></caption><graphic id="nlm-graphic-5" xlink:href="FEBS-283-1921-g002"></graphic></fig></sec><sec id="febs13711-sec-0004"><title>Structure of a protein complex of the VEGF‐A<sub>165</sub> C‐terminus and the NRP2 b1 domain</title><p>We also obtained a crystal structure of a protein complex of peptide EG00086 (Fig. <xref rid="febs13711-fig-0003" ref-type="fig">3</xref>A) bound to the b1 domain of NRP2. Peptide EG00086, corresponding to the C‐terminus of VEGF‐A<sub>165</sub> (residues 138–165), its binding affinity to NRP1 and NRP2, and its potency to act as inhibitor of VEGF‐promoted cellular adhesion have been previously described <xref rid="febs13711-bib-0029" ref-type="ref">29</xref>. The peptide corresponds to the C‐terminal half (one sub‐domain) of the HBD of VEGF‐A<sub>165</sub>. The small sub‐domain comprises short two‐stranded anti‐parallel β‐sheet stabilized by two disulfide bridges. The crystal structure of the complex between the NRP2 b1 domain and EG00086 was refined to 1.95 Å resolution, with four copies of EG00086‐bound NRP2 b1 in the asymmetric unit (Fig. <xref rid="febs13711-fig-0003" ref-type="fig">3</xref> and Table <xref rid="febs13711-tbl-0001" ref-type="table-wrap">1</xref>). In this structure, the C‐terminal arginine residue of the ligand EG00086 resides in the conserved binding pocket of the NRP2 b1 domain (Fig. <xref rid="febs13711-fig-0003" ref-type="fig">3</xref>C). The C‐terminal carboxylate forms hydrogen bonds with Ser349 and Thr352, while the Arg guanidinium group engages in ionic interaction with Asp323 at the base of the binding pocket. The aliphatic portion of the Arg side chain is nestled between Tyr356 and Tyr299, while the hydroxyl group of Tyr299 additionally interacts with EG00086 through a hydrogen bond with the backbone carbonyl oxygen of Pro163 of EG00086 (or residue 230 in a canonical full‐length VEGF‐A sequence, P15692‐1) (Fig. <xref rid="febs13711-fig-0003" ref-type="fig">3</xref>C). The NRP2 side chains contributing to tethering of the C‐terminal arginine are conserved in the NRP1 binding pocket <xref rid="febs13711-bib-0025" ref-type="ref">25</xref>, <xref rid="febs13711-bib-0026" ref-type="ref">26</xref>, <xref rid="febs13711-bib-0030" ref-type="ref">30</xref>, and have also been identified as being involved in binding of VEGF‐C within the structure of an NRP2 fusion protein in which the C‐terminus of the NRP2 b1b2 protein product is fused to the last five residues of proteolytically processed VEGF‐C <xref rid="febs13711-bib-0027" ref-type="ref">27</xref>. In our structure of the complex between NRP2 b1 domain and the EG00086 peptide, even though there is sufficient space within the crystal packing to accommodate the entire EG00086 molecule, a significant portion of the peptide appeared disordered, and, within the tetrameric structure, only up to eight amino acids of EG00086 per NRP2 binding site were modelled, with some of the side chains not visible in the electron density maps (Fig. <xref rid="febs13711-fig-0003" ref-type="fig">3</xref>C). While the C‐terminus of the peptide is well‐tethered to the neuropilin molecule, the rest of the structure displays multiple confirmations, giving rise to crystallographic disorder. Even though the peptide contains disulfide bonds, we were unable to model with confidence the adjacent β‐strand of the β‐hairpin structure. The structure of the complex implies that the main function of the HBD of VEGF may be to provide a specific but flexible connection between the two partner receptors: neuropilin and the VEGF receptor.</p><fig fig-type="Figure" xml:lang="en" id="febs13711-fig-0003" orientation="portrait" position="float"><label>Figure 3</label><caption><p>Structure of the <styled-content style="fixed-case">VEGF</styled-content>‐A<sub>165</sub>‐derived peptide bound to the <styled-content style="fixed-case">NRP</styled-content>2 b1 domain. (A) The amino acid sequences of the heparin binding domain of <styled-content style="fixed-case">VEGF</styled-content>‐A<sub>165</sub> and the related bicyclic peptide <styled-content style="fixed-case">EG</styled-content>00086. Disulfide links are indicated. The numbering corresponds to the full‐length <styled-content style="fixed-case">VEGF</styled-content>‐A form. (B) A maximum of eight C‐terminal residues of <styled-content style="fixed-case">EG</styled-content>00086 (sticks) were detected in the electron density maps of the molecular complex. The peptide binds to the canonical ligand binding site flanked by loops L1 and L3. (C) Detailed view of the interactions between the peptide and <styled-content style="fixed-case">NRP</styled-content>2. Relevant residues have been labelled, and the specific hydrogen bonds and charge–charge interactions are indicated by dashed lines. The 2 <italic>F</italic><sub>o</sub> – <italic>F</italic><sub>c</sub> electron density map (grey mesh) showed significant disorder for the peptide region; in this image, the associated electron density map is contoured at 0.6 σ.</p></caption><graphic id="nlm-graphic-7" xlink:href="FEBS-283-1921-g003"></graphic></fig><p>Intriguingly, in the EG00086‐bound NRP2 structure reported here and in the recently reported structure of the protein fusion between the NRP2 b1b2 segment and the C‐terminal pentapeptide of proteolytically processed VEGF‐C <xref rid="febs13711-bib-0027" ref-type="ref">27</xref>, both VEGF‐A<sub>165</sub> and VEGF‐C bind to the same NRP2 binding site, and the same set of hydrogen bonds and ionic interactions are involved in binding of the C‐terminal ligand residues. However, we observe differences in the direction of the C‐terminal VEGF‐A<sub>165</sub>‐related peptide between the structure reported here and that reported for the structure of the NRP1 b1–VEGF‐A<sub>165</sub>‐HBD fusion protein <xref rid="febs13711-bib-0026" ref-type="ref">26</xref>. However, in both structures, effects of crystal packing on positioning of the VEGF‐A<sub>165</sub> components of the complexes could not be completely excluded. Our previous isothermal titration calorimetry (ITC) measurements of the <italic>in vitro</italic> interaction between NRP1, NRP2 and EG00086 <xref rid="febs13711-bib-0029" ref-type="ref">29</xref>, and the crystal structures of the relevant complexes, do not clarify the molecular basis for the differences in biological activity. Considering that qualitatively the same set of key molecular interactions have been identified in all instances, it is possible that additional factors such as the energy of the additional hydrogen bonds within the NRP1/ligand and NRP2/ligand complexes, entropic changes due to side chains or solvent rearrangement within the ligand binding sites, or structural differences and post‐translational modifications elsewhere on the proteins contribute to the perceived differences in the ligand affinities of NRP1 versus NRP2.</p></sec><sec id="febs13711-sec-0005"><title>The zinc binding site in the NRP2 b1 domain is remote from the VEGF ligand binding site</title><p>Overlay of the two structures reported here shows that the main zinc binding site in the NRP2 b1 domain is remote from the VEGF ligand binding site (Fig. <xref rid="febs13711-fig-0004" ref-type="fig">4</xref>A), and is unlikely to directly affect ligand binding. Interestingly, although remote from the VEGF binding pocket, zinc ion binding site ‘1’ is located at the edge of the interface between the b1 and b2 domains (Fig. <xref rid="febs13711-fig-0004" ref-type="fig">4</xref>A). All neuropilin structures published so far <xref rid="febs13711-bib-0028" ref-type="ref">28</xref>, <xref rid="febs13711-bib-0030" ref-type="ref">30</xref>, <xref rid="febs13711-bib-0031" ref-type="ref">31</xref> that contain tandem b1b2 domains exhibit an extensive inter‐domain interface, providing fixed orientation of the two constituting domains. Binding of a zinc ion in that region may affect the interface and overall protein conformation and stability. Overlay of our structure with that of the b1b2 domain (PDB ID <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=2QQJ">2QQJ</ext-link>) shows that, in addition to the adjustments of the side‐chain positions required for ion binding, there is a significant effect on the position of backbone residues and temperature factors in this area (Fig. <xref rid="febs13711-fig-0004" ref-type="fig">4</xref>B). There is a localized backbone displacement of 1–2 Å for atoms of NRP2 residues in the regions spanning Tyr375–Asn380 and Ala400–Leu403. Binding of zinc also resulted in reduction of thermal motions of the corresponding atoms (Fig. <xref rid="febs13711-fig-0004" ref-type="fig">4</xref>B).</p><fig fig-type="Figure" xml:lang="en" id="febs13711-fig-0004" orientation="portrait" position="float"><label>Figure 4</label><caption><p>Zn<sup>2+</sup> ions bind remotely from the peptide ligand binding site on <styled-content style="fixed-case">NRP</styled-content>2. (A) Overlay of the two structures reported here (yellow and orange, EG00086 and Zn ion‐bound respectively) with the structure of the <styled-content style="fixed-case">NRP</styled-content>2 b1b2 domains (PDB ID <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=2QQJ">2QQJ</ext-link>) (grey) shows that the main zinc binding site is located near the interface of domains b1 and b2, remote from the ligand binding region. <styled-content style="fixed-case">EG</styled-content>00086 is shown using sticks representation, and the zinc ion is indicated by a grey sphere. (B) Close‐up view of the conformational changes associated with Zn<sup>2+</sup> binding. The zinc‐bound structure shown in yellow is compared to <styled-content style="fixed-case">NRP</styled-content>2 b1b2 domains (PDB ID <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=2QQJ">2QQJ</ext-link>), shown in grey. In this representation, the backbone thickness correlates with the values of the temperature factors for the corresponding backbone atoms.</p></caption><graphic id="nlm-graphic-9" xlink:href="FEBS-283-1921-g004"></graphic></fig><p>Zinc ion binding was further investigated using ITC. Consistent with our crystal structure, the binding curve of zinc ion titration into the wild‐type NRP2 b1 domain is fitted with three binding sites (Fig. <xref rid="febs13711-fig-0005" ref-type="fig">5</xref>). Our ITC experiments identified the highest‐affinity binding site (site ’1’), with a dissociation constant (<italic>K</italic><sub>D</sub>) of 1.1 μ<sc>m</sc>. This affinity is expected for a binding site with three side chains coordinating the ion, and is within the range of affinity constants for other physiologically relevant zinc‐binding proteins <xref rid="febs13711-bib-0032" ref-type="ref">32</xref>. The micromolar affinity of NRP2 for zinc suggests that, rather than having a structural role, this zinc binding site may have a regulatory function that is exhibited at times of fluctuating physiological zinc concentrations. The other two dissociation constants that were obtained, 11.9 and 149.9 μ<sc>m</sc>, indicate much weaker affinity and may not be of physiological importance. When histidine residues in site ‘1’ were mutated to alanines (His377Ala and His399Ala), the NRP2 b1 domain was still able to bind zinc, but the titration curve was consistent with the presence of two binding sites (Fig. <xref rid="febs13711-fig-0005" ref-type="fig">5</xref>) with dissociation constants of 20.7 and 143.5 μ<sc>m</sc>. The two zinc binding sites identified in the His377Ala/His399Ala NRP2 b1 variant have measured affinities comparable to those of the two weaker binding sites identified in wild‐type NRP2 b1. This observation confirms that histidine to alanine mutation of residues 377 and 399 results in abolition of zinc binding site ‘1’. ITC was also used to confirm that the His377Ala/His399Ala mutations did not affect the ligand binding potential of the resultant protein (Fig. <xref rid="febs13711-fig-0006" ref-type="fig">6</xref>), with EG00086 association constants of 1.07E5 ± 1.63E4 and 0.96E5 ± 1.68E4 <sc>m</sc><sup>−1</sup> for the wild‐type and mutant protein, respectively.</p><fig fig-type="Figure" xml:lang="en" id="febs13711-fig-0005" orientation="portrait" position="float"><label>Figure 5</label><caption><p>Mutation of residues His377 and His399 leads to a loss of <styled-content style="fixed-case">NRP</styled-content>2 b1 domain affinity for zinc. Isothermal titration of ZnCl<sub>2</sub> and site‐directed mutagenesis were used to confirm the role of residues His377 and His399 in ion binding. The panels on the left show the raw data (top) and the binding isotherm (bottom) for the wild‐type protein, while the panels on the right correspond to the double mutant <styled-content style="fixed-case">NRP</styled-content>2 b1b2 protein titrated with ZnCl<sub>2</sub>.</p></caption><graphic id="nlm-graphic-11" xlink:href="FEBS-283-1921-g005"></graphic></fig><fig fig-type="Figure" xml:lang="en" id="febs13711-fig-0006" orientation="portrait" position="float"><label>Figure 6</label><caption><p><styled-content style="fixed-case">ITC</styled-content> confirmed that mutation of His377 and His399 to Ala did not affect the interaction of the <styled-content style="fixed-case">NRP</styled-content>2 b1 domain with <styled-content style="fixed-case">EG</styled-content>00086. The panels on the left show the raw data (top) and the binding isotherm (bottom) for the wild‐type protein, while the panels on the right correspond to the double mutant <styled-content style="fixed-case">NRP</styled-content>2 b1b2 protein titrated with <styled-content style="fixed-case">EG</styled-content>00086.</p></caption><graphic id="nlm-graphic-13" xlink:href="FEBS-283-1921-g006"></graphic></fig></sec><sec id="febs13711-sec-0006"><title>NRP2 thermal stability is decreased in the presence of zinc</title><p>To further investigate the effect of zinc on NRP2, we performed a fluorescence‐based thermal denaturation assay, which supported our hypothesis that the zinc ion affects the stability of the NRP2 b1b2 protein. Using this rapid, qualitative assessment, we observed that, in the presence of zinc ions, there is a significant negative shift in thermal denaturation curves for all concentrations of Zn<sup>2+</sup> used. As an example of the effect, Fig. <xref rid="febs13711-fig-0007" ref-type="fig">7</xref> presents thermal denaturation curves showing that the melting temperature (<italic>T</italic><sub>m</sub>) for the NRP2 b1b2 protein decreased by 2 °C (<italic>T</italic><sub>m</sub> for NRP2 b1b2 = 44.5 °C; <italic>T</italic><sub>m</sub> for NRP2 b1b2‐Zn<sup>2+</sup> = 42.5 °C) in the presence of an equimolar amount of zinc. The crystal structure of the b1 domain revealed that Zn<sup>2+</sup> bound to site ‘1’ bridges the two structural loops that contain the Zn<sup>2+</sup>‐coordinating residues, thereby restricting their flexibility, as determined by the localized reduction in temperature factors. However, in the context of the b1b2 protein, this localized conformational distortion at the inter‐domain interface may contribute to overall loss of thermodynamic stability. The <italic>T</italic><sub>m</sub> decreased even further with an increase in zinc ion concentration (data not shown), most likely due to inter‐molecular zinc binding, causing aggregation and faster denaturation. Importantly, not only did the presence of an equimolar concentration of zinc result in NRP2 destabilization, the melting temperature of the histidine to alanine double mutant is itself 1 °C lower than that of wild‐type NRP2 b1b2 (Fig. <xref rid="febs13711-fig-0007" ref-type="fig">7</xref>), suggesting that the mutations at site 1 may disrupt the b1/b2 interface at some level, and decrease the overall stability of the variant protein. The effect of zinc in the context of the histidine to alanine double mutant of NRP2 b1b2 is a consequence of zinc ion binding in an inter‐molecular fashion, leading to protein aggregation and denaturation.</p><fig fig-type="Figure" xml:lang="en" id="febs13711-fig-0007" orientation="portrait" position="float"><label>Figure 7</label><caption><p>Zinc binding destabilizes <styled-content style="fixed-case">NRP</styled-content>2 b1b2 protein. The results of thermofluor assays for the wild‐type protein (top) and the mutant protein (bottom) in the presence of Zn<sup>2+</sup> and/or heparin demonstrate the protective function of heparin with respect to the protein instability introduced by zinc ion binding.</p></caption><graphic id="nlm-graphic-15" xlink:href="FEBS-283-1921-g007"></graphic></fig></sec><sec id="febs13711-sec-0007"><title>Heparin provides protection from zinc‐induced protein instability</title><p>Thermal shift assays have also shown that heparin provides protection from zinc‐induced thermal denaturation of NRP2 b1b2 (Fig. <xref rid="febs13711-fig-0007" ref-type="fig">7</xref>). It has been reported previously that heparin oligosaccharides bind NRP1 b1b2 and induce protein dimerization <xref rid="febs13711-bib-0030" ref-type="ref">30</xref>, and we used ITC to further examine the effect of heparin on the NRP2 b1b2 structure. When heparin oligosaccharides (20‐mer) were titrated into NRP2 b1b2, the ITC binding curve was consistent with two binding events characterized by dissociation constants of 0.33 and 5.81 μ<sc>m</sc> (Fig. <xref rid="febs13711-fig-0008" ref-type="fig">8</xref>A). The ITC curve also suggests that, in the initial stages of titration, both binding sites on the heparin molecule (higher‐ and lower‐affinity sites) are occupied by NRP2 b1b2, indicative of a stoichiometry of one molecule of heparin to two molecules of NRP2; but as the heparin concentration increases within the sample chamber, the higher‐affinity binding site is preferentially occupied until all of the NRP2 b1b2 molecules are heparin‐bound in a 1 : 1 stoichiometry. Even though heparin and NRP2 b1b2 form a 1 : 1 binding complex, heparin‐bound NRP2 b1b2 following ITC titration forms a dimer, as suggested by the size‐exclusion chromatography profile. In addition to inducing NRP2 b1b2 dimerization, heparin affects the stability of the NRP2 b1b2 protein, resulting in an increase in the <italic>T</italic><sub>m</sub> of heparin‐bound NRP2 b1b2 by 5.5 °C compared to NRP2 b1b2 alone (<italic>T</italic><sub>m</sub> = 50 °C and 44.5 °C, respectively) (Fig. <xref rid="febs13711-fig-0007" ref-type="fig">7</xref>). To assess the influence of heparin on zinc‐bound NRP2 b1b2 (<italic>T</italic><sub>m</sub> = 42.5 °C), we incubated a zinc/NRP2 b1b2 mixture in the presence of heparin for 30 min prior to the thermal shift assay. The presence of heparin effectively reversed the detrimental effect that zinc has on NRP2 b1b2 stability, with a <italic>T</italic><sub>m</sub> of 50 °C for heparin‐bound NRP2 b1b2 in the absence and presence of zinc (Fig. <xref rid="febs13711-fig-0007" ref-type="fig">7</xref>).</p><fig fig-type="Figure" xml:lang="en" id="febs13711-fig-0008" orientation="portrait" position="float"><label>Figure 8</label><caption><p>The putative heparin binding region on <styled-content style="fixed-case">NRP</styled-content>2 and zinc binding site ‘1’ are close to each other. (A) <styled-content style="fixed-case">ITC</styled-content> data for the interaction between heparin and <styled-content style="fixed-case">NRP</styled-content>2 b1b2 protein. The top panel shows raw data while the bottom panel shows the binding isotherm obtained by integrating peaks from the top panel. The curve is consistent with the occurrence of two binding events followed by <styled-content style="fixed-case">NRP</styled-content>2 dimerization. (B) The <styled-content style="fixed-case">NRP</styled-content>2 molecular surface electrostatic potential for the b1b2 domains (PDB ID <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=2QQJ">2QQJ</ext-link>) is overlaid with the molecular surface for the a1a2b1b2 ectopic region of <styled-content style="fixed-case">NRP</styled-content>2 (PDB ID <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=2QQK">2QQK</ext-link>), shown in grey. The zinc ion binding site in the <styled-content style="fixed-case">NRP</styled-content>2 structure reported here is indicated by a grey sphere, and the region of the positive electrostatic surface potential analogous to the <styled-content style="fixed-case">NRP</styled-content>1 heparin binding site is outlined using a dashed line.</p></caption><graphic id="nlm-graphic-17" xlink:href="FEBS-283-1921-g008"></graphic></fig><p>Based on the putative heparin binding site on NRP1 proposed by Vander Kooi <italic>et al</italic>. <xref rid="febs13711-bib-0030" ref-type="ref">30</xref>, we identified the corresponding region of the NRP2 b1b2 molecular surface that carries positive charge and thus may potentially be involved in heparin binding (Fig. <xref rid="febs13711-fig-0008" ref-type="fig">8</xref>B). Interestingly, the putative heparin binding region and zinc binding site ‘1’ are close to each other. The proximity of the two binding sites may explain, at least in part, the protective effect of heparin with respect to protein destabilization induced by zinc. It is also possible that zinc and oligosaccharides present in the extracellular matrix, although having opposite effects, are both involved in regulating the signalling function of NRP2.</p></sec></sec><sec id="febs13711-sec-0008"><title>Discussion</title><p>We have reported the first crystal structure of a complex between the peptide corresponding to the C‐terminus of VEGF‐A<sub>165</sub> and the b1 domain of NRP2. The structure suggests that the atomic details of direct molecular interactions between the b1 protein domain of NRP2 and the C‐terminal ends of VEGF isoforms A and C are similar, as seen from comparisons between the structure reported here (including VEGF‐A<sub>165</sub>) and that of the structure containing the C‐terminus of VEGF‐C <xref rid="febs13711-bib-0027" ref-type="ref">27</xref>. The level of structural similarity is somewhat surprising given that NRP1 and NRP2 have distinct physiological preferences for the growth factors, with VEGF‐A<sub>165</sub> being the main NRP1 ligand, and VEGF‐C signalling primarily through NRP2. Previously, it has been suggested that electrostatic steering provides the mechanism for ligand selectivity between neuropilins and various VEGF isoforms <xref rid="febs13711-bib-0026" ref-type="ref">26</xref>. In fact, comparison of the EG00086‐bound NRP2 b1 structure with that of the NRP1 b1–VEGF‐A<sub>165</sub>‐HBD fusion protein shows two significantly different positions of VEGF‐A<sub>165</sub>‐HBD with respect to the neuropilin b1 domain. The backbone of EG00086 is tilted away from the NRP2 molecule, indicating an almost 90° difference in the putative positioning of VEGF‐A<sub>165</sub>‐HBD compared to that suggested by Parker <italic>et al</italic>. for the NRP1 structure <xref rid="febs13711-bib-0026" ref-type="ref">26</xref>. The structure reported here supports the hypothesis that VEGF‐A<sub>165</sub> does not approach the NRP2 binding pocket in the same way as the binding pocket of NRP1 due to differences in the L1 loop of the binding pocket <xref rid="febs13711-bib-0026" ref-type="ref">26</xref>. However, it is possible that, in the context of the fusion protein NRP1 b1–VEGF‐A<sub>165</sub>‐HBD and/or under the specific crystallization conditions, VEGF‐A<sub>165</sub>‐HBD was constrained to adopt the observed orientation. It is probable that additional elements of the molecular repertoire, such as glycosylation and other post‐translational modifications (of either NRPs or VEGF molecules), may be important determinants of the ligand recognition mechanism of neuropilins and their physiological function in the context of cell‐surface signalling assemblies.</p><p>NRP2 is a key molecule in chemotactic migration of monocyte‐derived dendritic cells, and this function is dependent upon polysialic acid modification of NRP2 <xref rid="febs13711-bib-0033" ref-type="ref">33</xref>, <xref rid="febs13711-bib-0034" ref-type="ref">34</xref>, <xref rid="febs13711-bib-0035" ref-type="ref">35</xref>. Polysialylation is a rare post‐translational modification that has best been described in the context of the neural cell adhesion molecule. NRP1 is not modified by polysialylation; however, it has been shown that NRP1 may be modified by chondroitin sulfate attachment to Ser612, and that this modification results in reduced cell migration and invasiveness compared to cells expressing non‐glycosylated NRP1 <xref rid="febs13711-bib-0036" ref-type="ref">36</xref>, <xref rid="febs13711-bib-0037" ref-type="ref">37</xref>. Ser612 is located in the peptide linker region between the b2 domain and the membrane‐proximal c domain of NRP1. Similarly, mass spectrometry analysis of polysialylated NRP2 confirmed that polysialylation modification also occurs in the equivalent region between the NRP2 b2 and c domains <xref rid="febs13711-bib-0038" ref-type="ref">38</xref>. NRP2 enhances chemotaxis of dendritic cells towards the chemokine CCL21 in a polysialylation‐dependent fashion, directing dendritic cells to lymphoid organs for activation of naive T cells via antigen presentation.</p><p>Our structural studies additionally revealed a new feature of the NRP2 b1 domain structure, namely three Zn<sup>2+</sup> binding sites, of which the most biologically relevant involves coordination of a Zn<sup>2+</sup> ion with residues Tyr375, His377 and His399. The location of these residues, remote from the main VEGF binding pocket, suggests that Zn<sup>2+</sup> binding is unlikely to have a strong influence on NRP2–ligand interaction. ITC experiments have confirmed that mutations of His377 and His399 had no effect on ligand binding. However, mutation of His377 and His399 markedly decreased the thermal stability of the NRP2 b1 domain as revealed by thermal shift assays, an effect that was rescued by heparin binding to the NRP2 b1 domain. We predict that zinc binding to the b1/b2 inter‐domain interface would disrupt NRP2 structure and membrane organization, and possibly affect ligand recognition. However, in our experiments, binding of a model glycan (heparin) to NRP2 supported protein dimerization and protected NRP2 from zinc binding‐induced instability.</p><p>The physiological role of Zn<sup>2+</sup> binding to NRP2 is unclear, but, interestingly, NRP2 has been strongly implicated in maturation of dendritic cells, a process in which zinc homeostasis also plays a crucial role <xref rid="febs13711-bib-0039" ref-type="ref">39</xref>, <xref rid="febs13711-bib-0040" ref-type="ref">40</xref>, <xref rid="febs13711-bib-0041" ref-type="ref">41</xref>. Although the molecular mechanisms through which zinc affects maturation of dendritic cells are not well characterized, it has been shown that expression of molecules of the major histocompatibility complex on the plasma membrane correlates with a decrease in the expression of zinc transporters that move zinc into the cytosol, reduction of intracellular zinc, and an increase in the expression of zinc transporters that transfer ions in the opposite direction across the plasma membrane <xref rid="febs13711-bib-0039" ref-type="ref">39</xref>. Our structural identification and <italic>in vitro</italic> investigation of Zn<sup>2+</sup>/NRP2 interactions lay the foundation for further exploration of the effects of zinc homeostasis on neuropilin functions and ligand binding.</p></sec><sec id="febs13711-sec-0009"><title>Experimental procedures</title><sec id="febs13711-sec-0010"><title>Plasmids</title><p>Plasmids expressing recombinant NRP2 domains and VEGF‐A<sub>165</sub>‐HBD have been described previously <xref rid="febs13711-bib-0042" ref-type="ref">42</xref>. Wild‐type or mutant pET15b‐TEV:<italic>nrp2‐b1</italic>, pET15b‐TEV:<italic>nrp2‐b1b2</italic> constructs were transformed into Rosetta‐gami 2(DE3)pLysS cells (Novagen, Merck, Darmstadt, Germany), while pET14b:<italic>VEGF</italic>‐<italic>A</italic><sub><italic>165</italic></sub><italic>‐HBD</italic> was transformed into SHuffle cells (New England Biolabs, Ipswich, MA, USA).</p></sec><sec id="febs13711-sec-0011"><title>Site‐directed mutagenesis</title><p>The primers designed to introduce the H377A mutation were 5′‐CCTTGTGGTTTTTGCCAGCCCGGTACACCATC‐3′ and 5′CTGGATGGTGTACCGGGCTGGCAAAAACCAC‐3′. Those designed to introduce the H399A mutation were 5′‐GTCAGCAGTGGAGCGGCGAGCTTGTTCAGAAC‐3′ and 5′‐GGTTCTGAACAAGCTCGCCGCTCCACTGCTG‐3′. PCR was performed using Phusion high‐fidelity DNA polymerase (New England Biolabs) according to the manufacturer's instructions. In each reaction, 10 ng template (pET15b‐TEV:<italic>nrp2‐b1</italic> or pET15b‐TEV:<italic>nrp2‐b1b2</italic>) was added. The PCR product was treated with <italic>Dpn</italic>I at 37 °C for 1 h to cleave the mother template, followed by transformation of <italic>Escherichia coli</italic> DH5α cells (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA). The presence of the doubly mutated gene sequence (His377Ala/His399Ala) was confirmed by DNA sequencing (Source BioScience, Nottingham, UK).</p></sec><sec id="febs13711-sec-0012"><title>Expression and purification of recombinant proteins</title><p>To over‐express NRP2 b1, NRP2 b1b2 and VEGF‐A<sub>165</sub>‐HBD, 10 mL overnight cultures were transferred into 1 L of LB medium containing the appropriate antibiotics, and cells were grown at 37 °C until the attenuance at 600 nm reached 0.6. All cultures were grown in the presence of 100 ug·ml<sup>−1</sup> of ampicillin. In addition, tetracyclin at 12.5 ug·ml<sup>−1</sup> and chloramphenicol at 34 ug·ml<sup>−1</sup>, were used when growing Rosetta‐gami 2 (DE3)pLysS cells. Protein expression was induced by addition of isopropyl‐β‐<sc>d</sc>‐thiogalactopyranoside at a final concentration of 0.5 m<sc>m</sc>, and the cells were left overnight at 30 °C on an orbital shaker. Cells were harvested by centrifugation at 3993 <italic><bold>g</bold></italic> at 4 °C for 15 min, and washed once for 15 min with ice‐cold buffer A containing 50 m<sc>m</sc> Tris/HCl, pH 7.9, 300 m<sc>m</sc> NaCl and 30 m<sc>m</sc> imidazole. Cells were frozen and stored at −20 °C until used for protein purification.</p><p>Cells over‐expressing recombinant NRP2 b1 or b1b2 domains were resuspended in buffer A supplemented with EDTA‐free protease inhibitor (one tablet per 50 mL buffer, Roche) and lysed by sonication. The cell lysate was clarified by centrifugation at 39 100 <italic><bold>g</bold></italic> at 4 °C for 30 min. Soluble cell lysate was passed over a pre‐equilibrated nickel ion‐chelating affinity column (HisTrap column, GE Healthcare, Little Chalfont, UK). Tagged proteins were eluted at a flow rate of 1 mL·min<sup>−1</sup> using a linear gradient of buffer B (50 m<sc>m</sc> Tris/HCl, pH 7.9, 300 m<sc>m</sc> NaCl, 600 m<sc>m</sc> imidazole). Recombinant tobacco etch virus protease was mixed with eluted His‐tagged proteins, followed by dialysis against 4 L of 50 m<sc>m</sc> Tris/HCl, pH 8.0, 300 m<sc>m</sc> NaCl, 20 m<sc>m</sc> imidazole, 0.3 m<sc>m l</sc>‐cysteine and 3 m<sc>m l</sc>‐cystine at 4 °C overnight. Following dialysis, protein samples were incubated with pre‐equilibrated Ni‐NTA agarose (Qiagen, Hilden, Germany) for 30 min at 4 °C. Ni‐NTA agarose beads were pelleted by centrifugation at 3700 <italic><bold>g</bold></italic> at 4 °C for 10 min. The supernatant was collected and loaded onto a HiTrap Heparin HP column (GE Healthcare) pre‐equilibrated with 50 m<sc>m</sc> Tris/HCl, pH 7.9, 100 m<sc>m</sc> NaCl. Target protein was eluted at 1 mL·min<sup>−1</sup> using a linear gradient of high‐salt buffer comprising 50 m<sc>m</sc> Tris/HCl, pH 7.9, 2 <sc>m</sc> NaCl. Eluted recombinant neuropilin protein fractions were pooled, subjected to buffer exchange into 20 m<sc>m</sc> Tris/HCl, pH 7.9, 50 m<sc>m</sc> NaCl, and stored at 4 °C.</p><p>Cells over‐expressing recombinant VEGF‐A<sub>165</sub>‐HBD were lysed, and His‐tagged proteins were purified using a nickel‐chelating affinity column as described above. Thrombin was mixed with His‐tagged VEGF‐A<sub>165</sub>‐HBD, followed by dialysis against 4 L of 20 m<sc>m</sc> Tris pH 8.4, 150 m<sc>m</sc> NaCl, 2.5 m<sc>m</sc> CaCl<sub>2</sub>, at room temperature overnight. Tagged and untagged proteins were separated by incubation with Ni‐NTA agarose as described above. Untagged proteins were further purified using a HiTrap Heparin (HP) column pre‐equilibrated with 50 m<sc>m</sc> Tris/HCl, pH 7.9, 100 m<sc>m</sc> NaCl (GE Healthcare). Target protein was eluted at the flow rate of 1 mL·min<sup>−1</sup> using a linear gradient of high‐salt buffer containing 50 m<sc>m</sc> Tris/HCl, pH 7.9, 2 <sc>m</sc> NaCl. Fractions containing VEGF‐A<sub>165</sub>‐HBD were pooled, concentrated and loaded onto a pre‐equilibrated Superdex‐75 size‐exclusion column (GE Healthcare). VEGF‐A<sub>165</sub>‐HBD was eluted at 1 mL·min<sup>−1</sup> using buffer containing 50 m<sc>m</sc> Tris/HCl, pH 7.5, 100 m<sc>m</sc> NaCl. Samples were pooled, sparated into aliquots, and stored at −20 °C.</p></sec><sec id="febs13711-sec-0013"><title>Peptide synthesis</title><p>Synthesis of the bicyclic peptide, EG00086, containing the C‐terminal 28 amino acid residues of VEGF‐A<sub>165</sub> encoded by exons 7 and 8 (equivalent to VEGF‐A<sub>138–165</sub>), was performed as previously described <xref rid="febs13711-bib-0029" ref-type="ref">29</xref>.</p></sec><sec id="febs13711-sec-0014"><title>X‐ray crystallography</title><p>Purified NRP2 b1 domain at 10 mg·mL<sup>−1</sup> was crystallized in the presence of zinc sulfate. Protein was mixed with mother liquor (25% PEG 550 MME, 0.1 <sc>m</sc> MES, pH 6.5, 0.01 <sc>m</sc> ZnSO<sub>4</sub>) at a 1 : 1 ratio, and crystals were grown at 16 °C by the vapour diffusion method. X‐ray diffraction data were obtained at the European Synchrotron Radiation Facility (Grenoble, France) at 1.8 Å resolution, and belonged to space group C2221.</p><p>Formation of a complex of the NRP2 b1 domain and peptide EG00086 was confirmed by ITC, after which the sample was collected from the titration cell. The sample contained a 1 : 2 molar ratio of protein to ligand in 20 m<sc>m</sc> Tris/HCl, pH 7.9, 50 m<sc>m</sc> NaCl, and NRP2 b1 at a concentration of 10 mg·mL<sup>−1</sup>. The protein complex was mixed with mother liquor (10% PEG 20 000, 0.1 <sc>m</sc> bicine pH 9.0, 2% dioxane) at a 1 : 1 ratio, and crystals were grown at 16 °C by the vapour diffusion method. X‐ray diffraction data were collected at the Diamond Light Source (Didcot, UK) at 2 Å resolution, and belonged to space group C222<sub>1</sub> with cell dimensions of <italic>a</italic> = 110.25 Å, <italic>b</italic> = 139.52 Å, <italic>c</italic> = 106.04 Å, α = 90.00°, β = 90.00° and γ = 90.00°.</p><p>Data were merged and indexed using iMosflm <xref rid="febs13711-bib-0043" ref-type="ref">43</xref>, <xref rid="febs13711-bib-0044" ref-type="ref">44</xref>. The structure was solved by molecular replacement using <sc>phaser</sc><xref rid="febs13711-bib-0045" ref-type="ref">45</xref> with the apo NRP2 b1 domain (PDB ID <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=2QQJ">2QQJ</ext-link>) <xref rid="febs13711-bib-0028" ref-type="ref">28</xref>. Iterative rounds of refinement were performed using <sc>coot</sc><xref rid="febs13711-bib-0046" ref-type="ref">46</xref> and <sc>refmac5</sc><xref rid="febs13711-bib-0047" ref-type="ref">47</xref>, <xref rid="febs13711-bib-0048" ref-type="ref">48</xref>. CC<sub>½</sub> values were obtained from unmerged data using <sc>aimless</sc><xref rid="febs13711-bib-0049" ref-type="ref">49</xref>.</p></sec><sec id="febs13711-sec-0015"><title>Isothermal titration calorimetry</title><p>Wild‐type and mutants of the NRP2 b1 or b1b2 domains were dialysed in 20 m<sc>m</sc> Tris/HCl, pH 7.9, 50 m<sc>m</sc> NaCl at 4 °C overnight. Protein was loaded into the cell at 40–200 μ<sc>m</sc> concentration, while ZnCl<sub>2</sub> was loaded in the syringe at 1.5–5 m<sc>m</sc>. Zinc titration was performed at 21 °C using 98 injections in a MicroCal iTC200 system (GE Healthcare). EG00086 titration was performed at 15 °C with 19 injections using 40.3 μ<sc>m</sc> protein sample in the cell and 600 μ<sc>m</sc> EG00086 peptide in the syringe. VEGF‐A<sub>165</sub>‐HBD titration was performed at 10 °C with 19 injections, with 80 μ<sc>m</sc> protein sample in the cell and 2.1 m<sc>m</sc> VEGF‐A<sub>165</sub>‐HBD in the syringe. Titration of heparin oligosaccharides was performed at 21 °C with 39 injections of 300 μ<sc>m</sc> heparin oligosaccharides in the syringe and 36 μ<sc>m</sc> purified NRP2 b1b2 domain in the cell. Data were evaluated using <sc>origin</sc> version 7.0 software (OriginLab, Northampton, MA, USA) with the ITC plug‐in provided by the manufacturer.</p></sec><sec id="febs13711-sec-0016"><title>Thermofluor assay</title><p>The wild‐type and mutated NRP2 b1b2 domains (45 μ<sc>m</sc>) were mixed with 0, 10, 25, 45, 80, 150, 200 or 300 μ<sc>m</sc> ZnCl<sub>2</sub> in 20 m<sc>m</sc> Tris/HCl, pH 7.9, 50 m<sc>m</sc> NaCl, at room temperature for 30 min before mixing with SYPRO Orange protein gel stain (final dilution 1 : 1250; Life Technologies, Carlsbad, CA, USA). In the experiments including heparin, 45 μ<sc>m</sc> protein samples were mixed with 45 μ<sc>m</sc> ZnCl<sub>2</sub> at room temperature for 30 min before addition of 45 μ<sc>m</sc> heparin oligosaccharide dp20 (molecular mass 5750 Da; Iduron, Alderley Edge, UK). The samples were left at 4 °C for 30 min, and then mixed with the dye. The thermofluor assay was performed at 10–95 °C with a 0.5 °C step increment over 75 min using a MyiQ RT‐PCR instrument (Bio‐Rad, Hercules, CA, USA). Experiments were performed in triplicate. Data were evaluated using <sc>iq5</sc> software (Bio‐Rad).</p></sec><sec id="febs13711-sec-0017"><title>Accession numbers</title><p>The the EG00086 and Zn<sup>2+</sup>‐bound structures have been submitted to the Protein Data Bank under accession numbers <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5DN2">5DN2</ext-link> and <ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5DQ0">5DQ0</ext-link>, respectively.</p></sec></sec><sec id="febs13711-sec-0019"><title>Author contributions</title><p>Y.‐C.I.T., S.D., P.F. and I.Z. designed the research. Y.‐C.I.T. performed the majority of the experimental work. T.Y. performed part of the ITC experiments. R.R.R. and C.F. assisted in the refinement. Y.‐C.I.T., I.Z. and S.D. wrote the paper, and all authors revised the manuscript.</p></sec></body><back><ack id="febs13711-sec-0018"><title>Acknowledgements</title><p>We wish to acknowledge use of the Institute of Structural and Molecular Biology X‐ray crystallography facilities at the Institute of Structural and Molecular Biology and the laboratory manager Ambrose Cole for his contribution to maintaining crystallization robots and data collection. We thank Diamond Light Source for access to beamline I03. We also thank the European Synchrotron Radiation Facility for provision of synchrotron radiation facilities, and would like to thank their staff for assistance in using beamline ID23‐2. ITC data were obtained at the University College London School of Life and Medical Sciences ITC/CD facility. The project was funded primarily by British Heart Foundation research project grant PG/10/52/28448. C.F. and R.R.R. were funded by Magnus Life Science.</p></ack><ref-list content-type="cited-references" id="febs13711-bibl-0001"><title>References</title><ref id="febs13711-bib-0001"><label>1</label><mixed-citation publication-type="journal" id="febs13711-cit-0001"><string-name><surname>Chen</surname><given-names>H</given-names></string-name>, <string-name><surname>Chedotal</surname><given-names>A</given-names></string-name>, <string-name><surname>He</surname><given-names>Z</given-names></string-name>, <string-name><surname>Goodman</surname><given-names>CS</given-names></string-name> & <string-name><surname>Tessier‐Lavigne</surname><given-names>M</given-names></string-name> (<year>1997</year>) <article-title>Neuropilin‐2, a novel member of the neuropilin family, is a high affinity receptor for the semaphorins Sema E and Sema IV but not Sema III</article-title>. <source>Neuron</source><volume>19</volume>, <fpage>547</fpage>–<lpage>559</lpage>.<pub-id pub-id-type="pmid">9331348</pub-id></mixed-citation></ref><ref id="febs13711-bib-0002"><label>2</label><mixed-citation publication-type="journal" id="febs13711-cit-0002"><string-name><surname>Pellet‐Many</surname><given-names>C</given-names></string-name>, <string-name><surname>Frankel</surname><given-names>P</given-names></string-name>, <string-name><surname>Jia</surname><given-names>H</given-names></string-name> & <string-name><surname>Zachary</surname><given-names>I</given-names></string-name> (<year>2008</year>) <article-title>Neuropilins: structure, function and role in disease</article-title>. <source>Biochem J</source><volume>411</volume>, <fpage>211</fpage>–<lpage>226</lpage>.<pub-id pub-id-type="pmid">18363553</pub-id></mixed-citation></ref><ref id="febs13711-bib-0003"><label>3</label><mixed-citation publication-type="journal" id="febs13711-cit-0003"><string-name><surname>Djordjevic</surname><given-names>S</given-names></string-name> & <string-name><surname>Driscoll</surname><given-names>PC</given-names></string-name> (<year>2013</year>) <article-title>Targeting VEGF signalling via the neuropilin co‐receptor</article-title>. <source>Drug Discov Today</source><volume>18</volume>, <fpage>447</fpage>–<lpage>455</lpage>.<pub-id pub-id-type="pmid">23228652</pub-id></mixed-citation></ref><ref id="febs13711-bib-0004"><label>4</label><mixed-citation publication-type="journal" id="febs13711-cit-0004"><string-name><surname>Kitsukawa</surname><given-names>T</given-names></string-name>, <string-name><surname>Shimizu</surname><given-names>M</given-names></string-name>, <string-name><surname>Sanbo</surname><given-names>M</given-names></string-name>, <string-name><surname>Hirata</surname><given-names>T</given-names></string-name>, <string-name><surname>Taniguchi</surname><given-names>M</given-names></string-name>, <string-name><surname>Bekku</surname><given-names>Y</given-names></string-name>, <string-name><surname>Yagi</surname><given-names>T</given-names></string-name> & <string-name><surname>Fujisawa</surname><given-names>H</given-names></string-name> (<year>1997</year>) <article-title>Neuropilin–semaphorin III/D‐mediated chemorepulsive signals play a crucial role in peripheral nerve projection in mice</article-title>. <source>Neuron</source><volume>19</volume>, <fpage>995</fpage>–<lpage>1005</lpage>.<pub-id pub-id-type="pmid">9390514</pub-id></mixed-citation></ref><ref id="febs13711-bib-0005"><label>5</label><mixed-citation publication-type="journal" id="febs13711-cit-0005"><string-name><surname>Kawasaki</surname><given-names>T</given-names></string-name>, <string-name><surname>Kitsukawa</surname><given-names>T</given-names></string-name>, <string-name><surname>Bekku</surname><given-names>Y</given-names></string-name>, <string-name><surname>Matsuda</surname><given-names>Y</given-names></string-name>, <string-name><surname>Sanbo</surname><given-names>M</given-names></string-name>, <string-name><surname>Yagi</surname><given-names>T</given-names></string-name> & <string-name><surname>Fujisawa</surname><given-names>H</given-names></string-name> (<year>1999</year>) <article-title>A requirement for neuropilin‐1 in embryonic vessel formation</article-title>. <source>Development</source><volume>126</volume>, <fpage>4895</fpage>–<lpage>4902</lpage>.<pub-id pub-id-type="pmid">10518505</pub-id></mixed-citation></ref><ref id="febs13711-bib-0006"><label>6</label><mixed-citation publication-type="journal" id="febs13711-cit-0006"><string-name><surname>Gu</surname><given-names>C</given-names></string-name>, <string-name><surname>Rodriguez</surname><given-names>ER</given-names></string-name>, <string-name><surname>Reimert</surname><given-names>DV</given-names></string-name>, <string-name><surname>Shu</surname><given-names>T</given-names></string-name>, <string-name><surname>Fritzsch</surname><given-names>B</given-names></string-name>, <string-name><surname>Richards</surname><given-names>LJ</given-names></string-name>, <string-name><surname>Kolodkin</surname><given-names>AL</given-names></string-name> & <string-name><surname>Ginty</surname><given-names>DD</given-names></string-name> (<year>2003</year>) <article-title>Neuropilin‐1 conveys semaphorin and VEGF signaling during neural and cardiovascular development</article-title>. <source>Dev Cell</source><volume>5</volume>, <fpage>45</fpage>–<lpage>57</lpage>.<pub-id pub-id-type="pmid">12852851</pub-id></mixed-citation></ref><ref id="febs13711-bib-0007"><label>7</label><mixed-citation publication-type="journal" id="febs13711-cit-0007"><string-name><surname>Yuan</surname><given-names>L</given-names></string-name>, <string-name><surname>Moyon</surname><given-names>D</given-names></string-name>, <string-name><surname>Pardanaud</surname><given-names>L</given-names></string-name>, <string-name><surname>Breant</surname><given-names>C</given-names></string-name>, <string-name><surname>Karkkainen</surname><given-names>MJ</given-names></string-name>, <string-name><surname>Alitalo</surname><given-names>K</given-names></string-name> & <string-name><surname>Eichmann</surname><given-names>A</given-names></string-name> (<year>2002</year>) <article-title>Abnormal lymphatic vessel development in neuropilin‐2 mutant mice</article-title>. <source>Development</source><volume>129</volume>, <fpage>4797</fpage>–<lpage>4806</lpage>.<pub-id pub-id-type="pmid">12361971</pub-id></mixed-citation></ref><ref id="febs13711-bib-0008"><label>8</label><mixed-citation publication-type="journal" id="febs13711-cit-0008"><string-name><surname>Parker</surname><given-names>MW</given-names></string-name>, <string-name><surname>Guo</surname><given-names>HF</given-names></string-name>, <string-name><surname>Li</surname><given-names>X</given-names></string-name>, <string-name><surname>Linkugel</surname><given-names>AD</given-names></string-name> & <string-name><surname>Vander Kooi</surname><given-names>CW</given-names></string-name> (<year>2012</year>) <article-title>Function of members of the neuropilin family as essential pleiotropic cell surface receptors</article-title>. <source>Biochemistry</source><volume>51</volume>, <fpage>9437</fpage>–<lpage>9446</lpage>.<pub-id pub-id-type="pmid">23116416</pub-id></mixed-citation></ref><ref id="febs13711-bib-0009"><label>9</label><mixed-citation publication-type="journal" id="febs13711-cit-0009"><string-name><surname>Soker</surname><given-names>S</given-names></string-name>, <string-name><surname>Takashima</surname><given-names>S</given-names></string-name>, <string-name><surname>Miao</surname><given-names>HQ</given-names></string-name>, <string-name><surname>Neufeld</surname><given-names>G</given-names></string-name> & <string-name><surname>Klagsbrun</surname><given-names>M</given-names></string-name> (<year>1998</year>) <article-title>Neuropilin‐1 is expressed by endothelial and tumor cells as an isoform‐specific receptor for vascular endothelial growth factor</article-title>. <source>Cell</source><volume>92</volume>, <fpage>735</fpage>–<lpage>745</lpage>.<pub-id pub-id-type="pmid">9529250</pub-id></mixed-citation></ref><ref id="febs13711-bib-0010"><label>10</label><mixed-citation publication-type="journal" id="febs13711-cit-0010"><string-name><surname>Migdal</surname><given-names>M</given-names></string-name>, <string-name><surname>Huppertz</surname><given-names>B</given-names></string-name>, <string-name><surname>Tessler</surname><given-names>S</given-names></string-name>, <string-name><surname>Comforti</surname><given-names>A</given-names></string-name>, <string-name><surname>Shibuya</surname><given-names>M</given-names></string-name>, <string-name><surname>Reich</surname><given-names>R</given-names></string-name>, <string-name><surname>Baumann</surname><given-names>H</given-names></string-name> & <string-name><surname>Neufeld</surname><given-names>G</given-names></string-name> (<year>1998</year>) <article-title>Neuropilin‐1 is a placenta growth factor‐2 receptor</article-title>. <source>J Biol Chem</source><volume>273</volume>, <fpage>22272</fpage>–<lpage>22278</lpage>.<pub-id pub-id-type="pmid">9712843</pub-id></mixed-citation></ref><ref id="febs13711-bib-0011"><label>11</label><mixed-citation publication-type="journal" id="febs13711-cit-0011"><string-name><surname>Karpanen</surname><given-names>T</given-names></string-name>, <string-name><surname>Heckman</surname><given-names>CA</given-names></string-name>, <string-name><surname>Keskitalo</surname><given-names>S</given-names></string-name>, <string-name><surname>Jeltsch</surname><given-names>M</given-names></string-name>, <string-name><surname>Ollila</surname><given-names>H</given-names></string-name>, <string-name><surname>Neufeld</surname><given-names>G</given-names></string-name>, <string-name><surname>Tamagnone</surname><given-names>L</given-names></string-name> & <string-name><surname>Alitalo</surname><given-names>K</given-names></string-name> (<year>2006</year>) <article-title>Functional interaction of VEGF‐C and VEGF‐D with neuropilin receptors</article-title>. <source>FASEB J</source><volume>20</volume>, <fpage>1462</fpage>–<lpage>1472</lpage>.<pub-id pub-id-type="pmid">16816121</pub-id></mixed-citation></ref><ref id="febs13711-bib-0012"><label>12</label><mixed-citation publication-type="journal" id="febs13711-cit-0012"><string-name><surname>He</surname><given-names>Z</given-names></string-name> & <string-name><surname>Tessier‐Lavigne</surname><given-names>M</given-names></string-name> (<year>1997</year>) <article-title>Neuropilin is a receptor for the axonal chemorepellent semaphorin III</article-title>. <source>Cell</source><volume>90</volume>, <fpage>739</fpage>–<lpage>751</lpage>.<pub-id pub-id-type="pmid">9288753</pub-id></mixed-citation></ref><ref id="febs13711-bib-0013"><label>13</label><mixed-citation publication-type="journal" id="febs13711-cit-0013"><string-name><surname>Kolodkin</surname><given-names>AL</given-names></string-name>, <string-name><surname>Levengood</surname><given-names>DV</given-names></string-name>, <string-name><surname>Rowe</surname><given-names>EG</given-names></string-name>, <string-name><surname>Tai</surname><given-names>YT</given-names></string-name>, <string-name><surname>Giger</surname><given-names>RJ</given-names></string-name> & <string-name><surname>Ginty</surname><given-names>DD</given-names></string-name> (<year>1997</year>) <article-title>Neuropilin is a semaphorin III receptor</article-title>. <source>Cell</source><volume>90</volume>, <fpage>753</fpage>–<lpage>762</lpage>.<pub-id pub-id-type="pmid">9288754</pub-id></mixed-citation></ref><ref id="febs13711-bib-0014"><label>14</label><mixed-citation publication-type="journal" id="febs13711-cit-0014"><string-name><surname>Chen</surname><given-names>H</given-names></string-name>, <string-name><surname>He</surname><given-names>Z</given-names></string-name>, <string-name><surname>Bagri</surname><given-names>A</given-names></string-name> & <string-name><surname>Tessier‐Lavigne</surname><given-names>M</given-names></string-name> (<year>1998</year>) <article-title>Semaphorin–neuropilin interactions underlying sympathetic axon responses to class III semaphorins</article-title>. <source>Neuron</source><volume>21</volume>, <fpage>1283</fpage>–<lpage>1290</lpage>.<pub-id pub-id-type="pmid">9883722</pub-id></mixed-citation></ref><ref id="febs13711-bib-0015"><label>15</label><mixed-citation publication-type="journal" id="febs13711-cit-0015"><string-name><surname>Raper</surname><given-names>JA</given-names></string-name> (<year>2000</year>) <article-title>Semaphorins and their receptors in vertebrates and invertebrates</article-title>. <source>Curr Opin Neurobiol</source><volume>10</volume>, <fpage>88</fpage>–<lpage>94</lpage>.<pub-id pub-id-type="pmid">10679438</pub-id></mixed-citation></ref><ref id="febs13711-bib-0016"><label>16</label><mixed-citation publication-type="journal" id="febs13711-cit-0016"><string-name><surname>Hagberg</surname><given-names>CE</given-names></string-name>, <string-name><surname>Falkevall</surname><given-names>A</given-names></string-name>, <string-name><surname>Wang</surname><given-names>X</given-names></string-name>, <string-name><surname>Larsson</surname><given-names>E</given-names></string-name>, <string-name><surname>Huusko</surname><given-names>J</given-names></string-name>, <string-name><surname>Nilsson</surname><given-names>I</given-names></string-name>, <string-name><surname>van Meeteren</surname><given-names>LA</given-names></string-name>, <string-name><surname>Samen</surname><given-names>E</given-names></string-name>, <string-name><surname>Lu</surname><given-names>L</given-names></string-name>, <string-name><surname>Vanwildemeersch</surname><given-names>M</given-names></string-name><italic>et al</italic> (<year>2010</year>) <article-title>Vascular endothelial growth factor B controls endothelial fatty acid uptake</article-title>. <source>Nature</source><volume>464</volume>, <fpage>917</fpage>–<lpage>921</lpage>.<pub-id pub-id-type="pmid">20228789</pub-id></mixed-citation></ref><ref id="febs13711-bib-0017"><label>17</label><mixed-citation publication-type="journal" id="febs13711-cit-0017"><string-name><surname>Tran</surname><given-names>TS</given-names></string-name>, <string-name><surname>Rubio</surname><given-names>ME</given-names></string-name>, <string-name><surname>Clem</surname><given-names>RL</given-names></string-name>, <string-name><surname>Johnson</surname><given-names>D</given-names></string-name>, <string-name><surname>Case</surname><given-names>L</given-names></string-name>, <string-name><surname>Tessier‐Lavigne</surname><given-names>M</given-names></string-name>, <string-name><surname>Huganir</surname><given-names>RL</given-names></string-name>, <string-name><surname>Ginty</surname><given-names>DD</given-names></string-name> & <string-name><surname>Kolodkin</surname><given-names>AL</given-names></string-name> (<year>2009</year>) <article-title>Secreted semaphorins control spine distribution and morphogenesis in the postnatal CNS</article-title>. <source>Nature</source><volume>462</volume>, <fpage>1065</fpage>–<lpage>1069</lpage>.<pub-id pub-id-type="pmid">20010807</pub-id></mixed-citation></ref><ref id="febs13711-bib-0018"><label>18</label><mixed-citation publication-type="journal" id="febs13711-cit-0018"><string-name><surname>Zhang</surname><given-names>F</given-names></string-name>, <string-name><surname>Tang</surname><given-names>Z</given-names></string-name>, <string-name><surname>Hou</surname><given-names>X</given-names></string-name>, <string-name><surname>Lennartsson</surname><given-names>J</given-names></string-name>, <string-name><surname>Li</surname><given-names>Y</given-names></string-name>, <string-name><surname>Koch</surname><given-names>AW</given-names></string-name>, <string-name><surname>Scotney</surname><given-names>P</given-names></string-name>, <string-name><surname>Lee</surname><given-names>C</given-names></string-name>, <string-name><surname>Arjunan</surname><given-names>P</given-names></string-name>, <string-name><surname>Dong</surname><given-names>L</given-names></string-name><italic>et al</italic> (<year>2009</year>) <article-title>VEGF‐B is dispensable for blood vessel growth but critical for their survival, and VEGF‐B targeting inhibits pathological angiogenesis</article-title>. <source>Proc Natl Acad Sci USA</source><volume>106</volume>, <fpage>6152</fpage>–<lpage>6157</lpage>.<pub-id pub-id-type="pmid">19369214</pub-id></mixed-citation></ref><ref id="febs13711-bib-0019"><label>19</label><mixed-citation publication-type="journal" id="febs13711-cit-0019"><string-name><surname>Karkkainen</surname><given-names>MJ</given-names></string-name>, <string-name><surname>Saaristo</surname><given-names>A</given-names></string-name>, <string-name><surname>Jussila</surname><given-names>L</given-names></string-name>, <string-name><surname>Karila</surname><given-names>KA</given-names></string-name>, <string-name><surname>Lawrence</surname><given-names>EC</given-names></string-name>, <string-name><surname>Pajusola</surname><given-names>K</given-names></string-name>, <string-name><surname>Bueler</surname><given-names>H</given-names></string-name>, <string-name><surname>Eichmann</surname><given-names>A</given-names></string-name>, <string-name><surname>Kauppinen</surname><given-names>R</given-names></string-name>, <string-name><surname>Kettunen</surname><given-names>MI</given-names></string-name><italic>et al</italic> (<year>2001</year>) <article-title>A model for gene therapy of human hereditary lymphedema</article-title>. <source>Proc Natl Acad Sci USA</source><volume>98</volume>, <fpage>12677</fpage>–<lpage>12682</lpage>.<pub-id pub-id-type="pmid">11592985</pub-id></mixed-citation></ref><ref id="febs13711-bib-0020"><label>20</label><mixed-citation publication-type="journal" id="febs13711-cit-0020"><string-name><surname>Xu</surname><given-names>Y</given-names></string-name>, <string-name><surname>Yuan</surname><given-names>L</given-names></string-name>, <string-name><surname>Mak</surname><given-names>J</given-names></string-name>, <string-name><surname>Pardanaud</surname><given-names>L</given-names></string-name>, <string-name><surname>Caunt</surname><given-names>M</given-names></string-name>, <string-name><surname>Kasman</surname><given-names>I</given-names></string-name>, <string-name><surname>Larrivee</surname><given-names>B</given-names></string-name>, <string-name><surname>Del Toro</surname><given-names>R</given-names></string-name>, <string-name><surname>Suchting</surname><given-names>S</given-names></string-name>, <string-name><surname>Medvinsky</surname><given-names>A</given-names></string-name><italic>et al</italic> (<year>2010</year>) <article-title>Neuropilin‐2 mediates VEGF‐C‐induced lymphatic sprouting together with VEGFR3</article-title>. <source>J Cell Biol</source><volume>188</volume>, <fpage>115</fpage>–<lpage>130</lpage>.<pub-id pub-id-type="pmid">20065093</pub-id></mixed-citation></ref><ref id="febs13711-bib-0021"><label>21</label><mixed-citation publication-type="journal" id="febs13711-cit-0021"><string-name><surname>Soker</surname><given-names>S</given-names></string-name>, <string-name><surname>Miao</surname><given-names>HQ</given-names></string-name>, <string-name><surname>Nomi</surname><given-names>M</given-names></string-name>, <string-name><surname>Takashima</surname><given-names>S</given-names></string-name> & <string-name><surname>Klagsbrun</surname><given-names>M</given-names></string-name> (<year>2002</year>) <article-title>VEGF<sub>165</sub> mediates formation of complexes containing VEGFR‐2 and neuropilin‐1 that enhance VEGF<sub>165</sub>–receptor binding</article-title>. <source>J Cell Biochem</source><volume>85</volume>, <fpage>357</fpage>–<lpage>368</lpage>.<pub-id pub-id-type="pmid">11948691</pub-id></mixed-citation></ref><ref id="febs13711-bib-0022"><label>22</label><mixed-citation publication-type="journal" id="febs13711-cit-0022"><string-name><surname>Herzog</surname><given-names>B</given-names></string-name>, <string-name><surname>Pellet‐Many</surname><given-names>C</given-names></string-name>, <string-name><surname>Britton</surname><given-names>G</given-names></string-name>, <string-name><surname>Hartzoulakis</surname><given-names>B</given-names></string-name> & <string-name><surname>Zachary</surname><given-names>IC</given-names></string-name> (<year>2011</year>) <article-title>VEGF binding to NRP1 is essential for VEGF stimulation of endothelial cell migration, complex formation between NRP1 and VEGFR2, and signaling via FAK Tyr407 phosphorylation</article-title>. <source>Mol Biol Cell</source><volume>22</volume>, <fpage>2766</fpage>–<lpage>2776</lpage>.<pub-id pub-id-type="pmid">21653826</pub-id></mixed-citation></ref><ref id="febs13711-bib-0023"><label>23</label><mixed-citation publication-type="journal" id="febs13711-cit-0023"><string-name><surname>Muller</surname><given-names>YA</given-names></string-name>, <string-name><surname>Li</surname><given-names>B</given-names></string-name>, <string-name><surname>Christinger</surname><given-names>HW</given-names></string-name>, <string-name><surname>Wells</surname><given-names>JA</given-names></string-name>, <string-name><surname>Cunningham</surname><given-names>BC</given-names></string-name> & <string-name><surname>de Vos</surname><given-names>AM</given-names></string-name> (<year>1997</year>) <article-title>Vascular endothelial growth factor: crystal structure and functional mapping of the kinase domain receptor binding site</article-title>. <source>Proc Natl Acad Sci USA</source><volume>94</volume>, <fpage>7192</fpage>–<lpage>7197</lpage>.<pub-id pub-id-type="pmid">9207067</pub-id></mixed-citation></ref><ref id="febs13711-bib-0024"><label>24</label><mixed-citation publication-type="journal" id="febs13711-cit-0024"><string-name><surname>Fairbrother</surname><given-names>WJ</given-names></string-name>, <string-name><surname>Champe</surname><given-names>MA</given-names></string-name>, <string-name><surname>Christinger</surname><given-names>HW</given-names></string-name>, <string-name><surname>Keyt</surname><given-names>BA</given-names></string-name> & <string-name><surname>Starovasnik</surname><given-names>MA</given-names></string-name> (<year>1998</year>) <article-title>Solution structure of the heparin‐binding domain of vascular endothelial growth factor</article-title>. <source>Structure</source><volume>6</volume>, <fpage>637</fpage>–<lpage>648</lpage>.<pub-id pub-id-type="pmid">9634701</pub-id></mixed-citation></ref><ref id="febs13711-bib-0025"><label>25</label><mixed-citation publication-type="journal" id="febs13711-cit-0025"><string-name><surname>Jarvis</surname><given-names>A</given-names></string-name>, <string-name><surname>Allerston</surname><given-names>CK</given-names></string-name>, <string-name><surname>Jia</surname><given-names>H</given-names></string-name>, <string-name><surname>Herzog</surname><given-names>B</given-names></string-name>, <string-name><surname>Garza‐Garcia</surname><given-names>A</given-names></string-name>, <string-name><surname>Winfield</surname><given-names>N</given-names></string-name>, <string-name><surname>Ellard</surname><given-names>K</given-names></string-name>, <string-name><surname>Aqil</surname><given-names>R</given-names></string-name>, <string-name><surname>Lynch</surname><given-names>R</given-names></string-name>, <string-name><surname>Chapman</surname><given-names>C</given-names></string-name><italic>et al</italic> (<year>2010</year>) <article-title>Small molecule inhibitors of the neuropilin‐1 vascular endothelial growth factor A (VEGF‐A) interaction</article-title>. <source>J Med Chem</source><volume>53</volume>, <fpage>2215</fpage>–<lpage>2226</lpage>.<pub-id pub-id-type="pmid">20151671</pub-id></mixed-citation></ref><ref id="febs13711-bib-0026"><label>26</label><mixed-citation publication-type="journal" id="febs13711-cit-0026"><string-name><surname>Parker</surname><given-names>MW</given-names></string-name>, <string-name><surname>Xu</surname><given-names>P</given-names></string-name>, <string-name><surname>Li</surname><given-names>X</given-names></string-name> & <string-name><surname>Vander Kooi</surname><given-names>CW</given-names></string-name> (<year>2012</year>) <article-title>Structural basis for selective vascular endothelial growth factor‐A (VEGF‐A) binding to neuropilin‐1</article-title>. <source>J Biol Chem</source><volume>287</volume>, <fpage>11082</fpage>–<lpage>11089</lpage>.<pub-id pub-id-type="pmid">22318724</pub-id></mixed-citation></ref><ref id="febs13711-bib-0027"><label>27</label><mixed-citation publication-type="journal" id="febs13711-cit-0027"><string-name><surname>Parker</surname><given-names>MW</given-names></string-name>, <string-name><surname>Linkugel</surname><given-names>AD</given-names></string-name>, <string-name><surname>Goel</surname><given-names>HL</given-names></string-name>, <string-name><surname>Wu</surname><given-names>T</given-names></string-name>, <string-name><surname>Mercurio</surname><given-names>AM</given-names></string-name> & <string-name><surname>Vander Kooi</surname><given-names>CW</given-names></string-name> (<year>2015</year>) <article-title>Structural basis for VEGF‐C binding to neuropilin‐2 and sequestration by a soluble splice form</article-title>. <source>Structure</source><volume>23</volume>, <fpage>677</fpage>–<lpage>687</lpage>.<pub-id pub-id-type="pmid">25752543</pub-id></mixed-citation></ref><ref id="febs13711-bib-0028"><label>28</label><mixed-citation publication-type="journal" id="febs13711-cit-0028"><string-name><surname>Appleton</surname><given-names>BA</given-names></string-name>, <string-name><surname>Wu</surname><given-names>P</given-names></string-name>, <string-name><surname>Maloney</surname><given-names>J</given-names></string-name>, <string-name><surname>Yin</surname><given-names>J</given-names></string-name>, <string-name><surname>Liang</surname><given-names>WC</given-names></string-name>, <string-name><surname>Stawicki</surname><given-names>S</given-names></string-name>, <string-name><surname>Mortara</surname><given-names>K</given-names></string-name>, <string-name><surname>Bowman</surname><given-names>KK</given-names></string-name>, <string-name><surname>Elliott</surname><given-names>JM</given-names></string-name>, <string-name><surname>Desmarais</surname><given-names>W</given-names></string-name><italic>et al</italic> (<year>2007</year>) <article-title>Structural studies of neuropilin/antibody complexes provide insights into semaphorin and VEGF binding</article-title>. <source>EMBO J</source><volume>26</volume>, <fpage>4902</fpage>–<lpage>4912</lpage>.<pub-id pub-id-type="pmid">17989695</pub-id></mixed-citation></ref><ref id="febs13711-bib-0029"><label>29</label><mixed-citation publication-type="journal" id="febs13711-cit-0029"><string-name><surname>Jia</surname><given-names>H</given-names></string-name>, <string-name><surname>Aqil</surname><given-names>R</given-names></string-name>, <string-name><surname>Cheng</surname><given-names>L</given-names></string-name>, <string-name><surname>Chapman</surname><given-names>C</given-names></string-name>, <string-name><surname>Shaikh</surname><given-names>S</given-names></string-name>, <string-name><surname>Jarvis</surname><given-names>A</given-names></string-name>, <string-name><surname>Chan</surname><given-names>AW</given-names></string-name>, <string-name><surname>Hartzoulakis</surname><given-names>B</given-names></string-name>, <string-name><surname>Evans</surname><given-names>IM</given-names></string-name>, <string-name><surname>Frolov</surname><given-names>A</given-names></string-name><italic>et al</italic> (<year>2014</year>) <article-title>N‐terminal modification of VEGF‐A C terminus‐derived peptides delineates structural features involved in neuropilin‐1 binding and functional activity</article-title>. <source>ChemBioChem</source><volume>15</volume>, <fpage>1161</fpage>–<lpage>1170</lpage>.<pub-id pub-id-type="pmid">24771685</pub-id></mixed-citation></ref><ref id="febs13711-bib-0030"><label>30</label><mixed-citation publication-type="journal" id="febs13711-cit-0030"><string-name><surname>Vander Kooi</surname><given-names>CW</given-names></string-name>, <string-name><surname>Jusino</surname><given-names>MA</given-names></string-name>, <string-name><surname>Perman</surname><given-names>B</given-names></string-name>, <string-name><surname>Neau</surname><given-names>DB</given-names></string-name>, <string-name><surname>Bellamy</surname><given-names>HD</given-names></string-name> & <string-name><surname>Leahy</surname><given-names>DJ</given-names></string-name> (<year>2007</year>) <article-title>Structural basis for ligand and heparin binding to neuropilin B domains</article-title>. <source>Proc Natl Acad Sci USA</source><volume>104</volume>, <fpage>6152</fpage>–<lpage>6157</lpage>.<pub-id pub-id-type="pmid">17405859</pub-id></mixed-citation></ref><ref id="febs13711-bib-0031"><label>31</label><mixed-citation publication-type="journal" id="febs13711-cit-0031"><string-name><surname>Janssen</surname><given-names>BJ</given-names></string-name>, <string-name><surname>Malinauskas</surname><given-names>T</given-names></string-name>, <string-name><surname>Weir</surname><given-names>GA</given-names></string-name>, <string-name><surname>Cader</surname><given-names>MZ</given-names></string-name>, <string-name><surname>Siebold</surname><given-names>C</given-names></string-name> & <string-name><surname>Jones</surname><given-names>EY</given-names></string-name> (<year>2012</year>) <article-title>Neuropilins lock secreted semaphorins onto plexins in a ternary signaling complex</article-title>. <source>Nat Struct Mol Biol</source><volume>19</volume>, <fpage>1293</fpage>–<lpage>1299</lpage>.<pub-id pub-id-type="pmid">23104057</pub-id></mixed-citation></ref><ref id="febs13711-bib-0032"><label>32</label><mixed-citation publication-type="journal" id="febs13711-cit-0032"><string-name><surname>Kochanczyk</surname><given-names>T</given-names></string-name>, <string-name><surname>Drozd</surname><given-names>A</given-names></string-name> & <string-name><surname>Krezel</surname><given-names>A</given-names></string-name> (<year>2015</year>) <article-title>Relationship between the architecture of zinc coordination and zinc binding affinity in proteins – insights into zinc regulation</article-title>. <source>Metallomics</source><volume>7</volume>, <fpage>244</fpage>–<lpage>257</lpage>.<pub-id pub-id-type="pmid">25255078</pub-id></mixed-citation></ref><ref id="febs13711-bib-0033"><label>33</label><mixed-citation publication-type="journal" id="febs13711-cit-0033"><string-name><surname>Bax</surname><given-names>M</given-names></string-name>, <string-name><surname>van Vliet</surname><given-names>SJ</given-names></string-name>, <string-name><surname>Litjens</surname><given-names>M</given-names></string-name>, <string-name><surname>Garcia‐Vallejo</surname><given-names>JJ</given-names></string-name> & <string-name><surname>van Kooyk</surname><given-names>Y</given-names></string-name> (<year>2009</year>) <article-title>Interaction of polysialic acid with CCL21 regulates the migratory capacity of human dendritic cells</article-title>. <source>PLoS One</source><volume>4</volume>, <fpage>e6987</fpage>.<pub-id pub-id-type="pmid">19750015</pub-id></mixed-citation></ref><ref id="febs13711-bib-0034"><label>34</label><mixed-citation publication-type="journal" id="febs13711-cit-0034"><string-name><surname>Rey‐Gallardo</surname><given-names>A</given-names></string-name>, <string-name><surname>Escribano</surname><given-names>C</given-names></string-name>, <string-name><surname>Delgado‐Martin</surname><given-names>C</given-names></string-name>, <string-name><surname>Rodriguez‐Fernandez</surname><given-names>JL</given-names></string-name>, <string-name><surname>Gerardy‐Schahn</surname><given-names>R</given-names></string-name>, <string-name><surname>Rutishauser</surname><given-names>U</given-names></string-name>, <string-name><surname>Corbi</surname><given-names>AL</given-names></string-name> & <string-name><surname>Vega</surname><given-names>MA</given-names></string-name> (<year>2010</year>) <article-title>Polysialylated neuropilin‐2 enhances human dendritic cell migration through the basic C‐terminal region of CCL21</article-title>. <source>Glycobiology</source><volume>20</volume>, <fpage>1139</fpage>–<lpage>1146</lpage>.<pub-id pub-id-type="pmid">20488940</pub-id></mixed-citation></ref><ref id="febs13711-bib-0035"><label>35</label><mixed-citation publication-type="journal" id="febs13711-cit-0035"><string-name><surname>Rey‐Gallardo</surname><given-names>A</given-names></string-name>, <string-name><surname>Delgado‐Martin</surname><given-names>C</given-names></string-name>, <string-name><surname>Gerardy‐Schahn</surname><given-names>R</given-names></string-name>, <string-name><surname>Rodriguez‐Fernandez</surname><given-names>JL</given-names></string-name> & <string-name><surname>Vega</surname><given-names>MA</given-names></string-name> (<year>2011</year>) <article-title>Polysialic acid is required for neuropilin‐2a/b‐mediated control of CCL21‐driven chemotaxis of mature dendritic cells and for their migration <italic>in vivo</italic></article-title>. <source>Glycobiology</source><volume>21</volume>, <fpage>655</fpage>–<lpage>662</lpage>.<pub-id pub-id-type="pmid">21199821</pub-id></mixed-citation></ref><ref id="febs13711-bib-0036"><label>36</label><mixed-citation publication-type="journal" id="febs13711-cit-0036"><string-name><surname>Shintani</surname><given-names>Y</given-names></string-name>, <string-name><surname>Takashima</surname><given-names>S</given-names></string-name>, <string-name><surname>Asano</surname><given-names>Y</given-names></string-name>, <string-name><surname>Kato</surname><given-names>H</given-names></string-name>, <string-name><surname>Liao</surname><given-names>Y</given-names></string-name>, <string-name><surname>Yamazaki</surname><given-names>S</given-names></string-name>, <string-name><surname>Tsukamoto</surname><given-names>O</given-names></string-name>, <string-name><surname>Seguchi</surname><given-names>O</given-names></string-name>, <string-name><surname>Yamamoto</surname><given-names>H</given-names></string-name>, <string-name><surname>Fukushima</surname><given-names>T</given-names></string-name><italic>et al</italic> (<year>2006</year>) <article-title>Glycosaminoglycan modification of neuropilin‐1 modulates VEGFR2 signaling</article-title>. <source>EMBO J</source><volume>25</volume>, <fpage>3045</fpage>–<lpage>3055</lpage>.<pub-id pub-id-type="pmid">16763549</pub-id></mixed-citation></ref><ref id="febs13711-bib-0037"><label>37</label><mixed-citation publication-type="journal" id="febs13711-cit-0037"><string-name><surname>Frankel</surname><given-names>P</given-names></string-name>, <string-name><surname>Pellet‐Many</surname><given-names>C</given-names></string-name>, <string-name><surname>Lehtolainen</surname><given-names>P</given-names></string-name>, <string-name><surname>D'Abaco</surname><given-names>GM</given-names></string-name>, <string-name><surname>Tickner</surname><given-names>ML</given-names></string-name>, <string-name><surname>Cheng</surname><given-names>L</given-names></string-name> & <string-name><surname>Zachary</surname><given-names>IC</given-names></string-name> (<year>2008</year>) <article-title>Chondroitin sulphate‐modified neuropilin 1 is expressed in human tumour cells and modulates 3D invasion in the U87MG human glioblastoma cell line through a p130Cas‐mediated pathway</article-title>. <source>EMBO Rep</source><volume>9</volume>, <fpage>983</fpage>–<lpage>989</lpage>.<pub-id pub-id-type="pmid">18704117</pub-id></mixed-citation></ref><ref id="febs13711-bib-0038"><label>38</label><mixed-citation publication-type="journal" id="febs13711-cit-0038"><string-name><surname>Rollenhagen</surname><given-names>M</given-names></string-name>, <string-name><surname>Buettner</surname><given-names>FF</given-names></string-name>, <string-name><surname>Reismann</surname><given-names>M</given-names></string-name>, <string-name><surname>Jirmo</surname><given-names>AC</given-names></string-name>, <string-name><surname>Grove</surname><given-names>M</given-names></string-name>, <string-name><surname>Behrens</surname><given-names>GM</given-names></string-name>, <string-name><surname>Gerardy‐Schahn</surname><given-names>R</given-names></string-name>, <string-name><surname>Hanisch</surname><given-names>FG</given-names></string-name> & <string-name><surname>Muhlenhoff</surname><given-names>M</given-names></string-name> (<year>2013</year>) <article-title>Polysialic acid on neuropilin‐2 is exclusively synthesized by the polysialyltransferase ST8SiaIV and attached to mucin‐type <italic>O</italic>‐glycans located between the b2 and c domain</article-title>. <source>J Biol Chem</source><volume>288</volume>, <fpage>22880</fpage>–<lpage>22892</lpage>.<pub-id pub-id-type="pmid">23801331</pub-id></mixed-citation></ref><ref id="febs13711-bib-0039"><label>39</label><mixed-citation publication-type="journal" id="febs13711-cit-0039"><string-name><surname>Kitamura</surname><given-names>H</given-names></string-name>, <string-name><surname>Morikawa</surname><given-names>H</given-names></string-name>, <string-name><surname>Kamon</surname><given-names>H</given-names></string-name>, <string-name><surname>Iguchi</surname><given-names>M</given-names></string-name>, <string-name><surname>Hojyo</surname><given-names>S</given-names></string-name>, <string-name><surname>Fukada</surname><given-names>T</given-names></string-name>, <string-name><surname>Yamashita</surname><given-names>S</given-names></string-name>, <string-name><surname>Kaisho</surname><given-names>T</given-names></string-name>, <string-name><surname>Akira</surname><given-names>S</given-names></string-name>, <string-name><surname>Murakami</surname><given-names>M</given-names></string-name><italic>et al</italic> (<year>2006</year>) <article-title>Toll‐like receptor‐mediated regulation of zinc homeostasis influences dendritic cell function</article-title>. <source>Nat Immunol</source><volume>7</volume>, <fpage>971</fpage>–<lpage>977</lpage>.<pub-id pub-id-type="pmid">16892068</pub-id></mixed-citation></ref><ref id="febs13711-bib-0040"><label>40</label><mixed-citation publication-type="journal" id="febs13711-cit-0040"><string-name><surname>John</surname><given-names>E</given-names></string-name>, <string-name><surname>Laskow</surname><given-names>TC</given-names></string-name>, <string-name><surname>Buchser</surname><given-names>WJ</given-names></string-name>, <string-name><surname>Pitt</surname><given-names>BR</given-names></string-name>, <string-name><surname>Basse</surname><given-names>PH</given-names></string-name>, <string-name><surname>Butterfield</surname><given-names>LH</given-names></string-name>, <string-name><surname>Kalinski</surname><given-names>P</given-names></string-name> & <string-name><surname>Lotze</surname><given-names>MT</given-names></string-name> (<year>2010</year>) <article-title>Zinc in innate and adaptive tumor immunity</article-title>. <source>J Transl Med</source><volume>8</volume>, <fpage>118</fpage>.<pub-id pub-id-type="pmid">21087493</pub-id></mixed-citation></ref><ref id="febs13711-bib-0041"><label>41</label><mixed-citation publication-type="journal" id="febs13711-cit-0041"><string-name><surname>Rink</surname><given-names>L</given-names></string-name> & <string-name><surname>Haase</surname><given-names>H</given-names></string-name> (<year>2007</year>) <article-title>Zinc homeostasis and immunity</article-title>. <source>Trends Immunol</source><volume>28</volume>, <fpage>1</fpage>–<lpage>4</lpage>.<pub-id pub-id-type="pmid">17126599</pub-id></mixed-citation></ref><ref id="febs13711-bib-0042"><label>42</label><mixed-citation publication-type="journal" id="febs13711-cit-0042"><string-name><surname>Seyedarabi</surname><given-names>A</given-names></string-name>, <string-name><surname>Cheng</surname><given-names>L</given-names></string-name>, <string-name><surname>Zachary</surname><given-names>I</given-names></string-name> & <string-name><surname>Djordjevic</surname><given-names>S</given-names></string-name> (<year>2013</year>) <article-title>Production of soluble human vascular endothelial growth factor VEGF‐A<sub>165</sub>‐heparin binding domain in <italic>Escherichia coli</italic></article-title>. <source>PLoS One</source><volume>8</volume>, <fpage>e55690</fpage>.<pub-id pub-id-type="pmid">23409021</pub-id></mixed-citation></ref><ref id="febs13711-bib-0043"><label>43</label><mixed-citation publication-type="journal" id="febs13711-cit-0043"><string-name><surname>Powell</surname><given-names>HR</given-names></string-name>, <string-name><surname>Johnson</surname><given-names>O</given-names></string-name> & <string-name><surname>Leslie</surname><given-names>AG</given-names></string-name> (<year>2013</year>) <article-title>Autoindexing diffraction images with iMosflm</article-title>. <source>Acta Crystallogr D</source><volume>69</volume>, <fpage>1195</fpage>–<lpage>1203</lpage>.<pub-id pub-id-type="pmid">23793145</pub-id></mixed-citation></ref><ref id="febs13711-bib-0044"><label>44</label><mixed-citation publication-type="book" id="febs13711-cit-0044"><string-name><surname>Leslie</surname><given-names>AW</given-names></string-name> & <string-name><surname>Powell</surname><given-names>H</given-names></string-name> (<year>2007</year>) <chapter-title>Processing diffraction data with mosflm</chapter-title> In <source>Evolving Methods for Macromolecular Crystallography</source> (<person-group person-group-type="editor"><name><surname>Read</surname><given-names>R</given-names></name></person-group> & <person-group person-group-type="editor"><name><surname>Sussman</surname><given-names>J</given-names></name></person-group>, eds), pp. <fpage>41</fpage>–<lpage>51</lpage>. <publisher-name>Springer</publisher-name>, <publisher-loc>Dordrecht, The Netherlands</publisher-loc>.</mixed-citation></ref><ref id="febs13711-bib-0045"><label>45</label><mixed-citation publication-type="journal" id="febs13711-cit-0045"><string-name><surname>McCoy</surname><given-names>AJ</given-names></string-name>, <string-name><surname>Grosse‐Kunstleve</surname><given-names>RW</given-names></string-name>, <string-name><surname>Adams</surname><given-names>PD</given-names></string-name>, <string-name><surname>Winn</surname><given-names>MD</given-names></string-name>, <string-name><surname>Storoni</surname><given-names>LC</given-names></string-name> & <string-name><surname>Read</surname><given-names>RJ</given-names></string-name> (<year>2007</year>) <article-title>Phaser crystallographic software</article-title>. <source>J Appl Crystallogr</source><volume>40</volume>, <fpage>658</fpage>–<lpage>674</lpage>.<pub-id pub-id-type="pmid">19461840</pub-id></mixed-citation></ref><ref id="febs13711-bib-0046"><label>46</label><mixed-citation publication-type="journal" id="febs13711-cit-0046"><string-name><surname>Emsley</surname><given-names>P</given-names></string-name> & <string-name><surname>Cowtan</surname><given-names>K</given-names></string-name> (<year>2004</year>) <article-title>Coot: model‐building tools for molecular graphics</article-title>. <source>Acta Crystallogr D</source><volume>60</volume>, <fpage>2126</fpage>–<lpage>2132</lpage>.<pub-id pub-id-type="pmid">15572765</pub-id></mixed-citation></ref><ref id="febs13711-bib-0047"><label>47</label><mixed-citation publication-type="journal" id="febs13711-cit-0047"><string-name><surname>Murshudov</surname><given-names>GN</given-names></string-name>, <string-name><surname>Vagin</surname><given-names>AA</given-names></string-name> & <string-name><surname>Dodson</surname><given-names>EJ</given-names></string-name> (<year>1997</year>) <article-title>Refinement of macromolecular structures by the maximum‐likelihood method</article-title>. <source>Acta Crystallogr D</source><volume>53</volume>, <fpage>240</fpage>–<lpage>255</lpage>.<pub-id pub-id-type="pmid">15299926</pub-id></mixed-citation></ref><ref id="febs13711-bib-0048"><label>48</label><mixed-citation publication-type="journal" id="febs13711-cit-0048"><string-name><surname>Vagin</surname><given-names>AA</given-names></string-name>, <string-name><surname>Steiner</surname><given-names>RA</given-names></string-name>, <string-name><surname>Lebedev</surname><given-names>AA</given-names></string-name>, <string-name><surname>Potterton</surname><given-names>L</given-names></string-name>, <string-name><surname>McNicholas</surname><given-names>S</given-names></string-name>, <string-name><surname>Long</surname><given-names>F</given-names></string-name> & <string-name><surname>Murshudov</surname><given-names>GN</given-names></string-name> (<year>2004</year>) <article-title>REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use</article-title>. <source>Acta Crystallogr D</source><volume>60</volume>, <fpage>2184</fpage>–<lpage>2195</lpage>.<pub-id pub-id-type="pmid">15572771</pub-id></mixed-citation></ref><ref id="febs13711-bib-0049"><label>49</label><mixed-citation publication-type="journal" id="febs13711-cit-0049"><string-name><surname>Evans</surname><given-names>PR</given-names></string-name> & <string-name><surname>Murshudov</surname><given-names>GN</given-names></string-name> (<year>2013</year>) <article-title>How good are my data and what is the resolution?</article-title><source>Acta Crystallogr D</source><volume>69</volume>, <fpage>1204</fpage>–<lpage>1214</lpage>.<pub-id pub-id-type="pmid">23793146</pub-id></mixed-citation></ref></ref-list></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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