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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Crystal Structure of Malaria Parasite Nucleosome Assembly
Protein</title><author><name sortKey="Gill, Jasmita" sort="Gill, Jasmita" uniqKey="Gill J" first="Jasmita" last="Gill">Jasmita Gill</name><affiliation><nlm:aff id="N0x1ca9390N0x43159b8">Structural and Computational Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Road, New Delhi 110067, India, the</nlm:aff></affiliation></author><author><name sortKey="Yogavel, Manickam" sort="Yogavel, Manickam" uniqKey="Yogavel M" first="Manickam" last="Yogavel">Manickam Yogavel</name><affiliation><nlm:aff id="N0x1ca9390N0x43159b8">Structural and Computational Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Road, New Delhi 110067, India, the</nlm:aff></affiliation></author><author><name sortKey="Kumar, Anuj" sort="Kumar, Anuj" uniqKey="Kumar A" first="Anuj" last="Kumar">Anuj Kumar</name><affiliation><nlm:aff id="N0x1ca9390N0x43159b8">Structural and Computational Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Road, New Delhi 110067, India, the</nlm:aff></affiliation></author><author><name sortKey="Belrhali, Hassan" sort="Belrhali, Hassan" uniqKey="Belrhali H" first="Hassan" last="Belrhali">Hassan Belrhali</name><affiliation><nlm:aff id="N0x1ca9390N0x43159b8">European Molecular Biology Laboratory, 6 Rue Jules Horowitz, BP 181, F-38042 Grenoble Cédex 9, France, the</nlm:aff></affiliation></author><author><name sortKey="Jain, S K" sort="Jain, S K" uniqKey="Jain S" first="S. K." last="Jain">S. K. Jain</name><affiliation><nlm:aff id="N0x1ca9390N0x43159b8">Department of Biotechnology, Hamdard University, Hamdard Nagar, New Delhi 110062, India, and the</nlm:aff></affiliation></author><author><name sortKey="Rug, Melanie" sort="Rug, Melanie" uniqKey="Rug M" first="Melanie" last="Rug">Melanie Rug</name><affiliation><nlm:aff id="N0x1ca9390N0x43159b8">Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia</nlm:aff></affiliation></author><author><name sortKey="Brown, Monica" sort="Brown, Monica" uniqKey="Brown M" first="Monica" last="Brown">Monica Brown</name><affiliation><nlm:aff id="N0x1ca9390N0x43159b8">Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia</nlm:aff></affiliation></author><author><name sortKey="Maier, Alexander G" sort="Maier, Alexander G" uniqKey="Maier A" first="Alexander G." last="Maier">Alexander G. Maier</name><affiliation><nlm:aff id="N0x1ca9390N0x43159b8">Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia</nlm:aff></affiliation></author><author><name sortKey="Sharma, Amit" sort="Sharma, Amit" uniqKey="Sharma A" first="Amit" last="Sharma">Amit Sharma</name><affiliation><nlm:aff id="N0x1ca9390N0x43159b8">Structural and Computational Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Road, New Delhi 110067, India, the</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">19176479</idno><idno type="pmc">2665062</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2665062</idno><idno type="RBID">PMC:2665062</idno><idno type="doi">10.1074/jbc.M808633200</idno><date when="2009">2009</date><idno type="wicri:Area/Pmc/Corpus">001117</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">001117</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Crystal Structure of Malaria Parasite Nucleosome Assembly
Protein</title><author><name sortKey="Gill, Jasmita" sort="Gill, Jasmita" uniqKey="Gill J" first="Jasmita" last="Gill">Jasmita Gill</name><affiliation><nlm:aff id="N0x1ca9390N0x43159b8">Structural and Computational Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Road, New Delhi 110067, India, the</nlm:aff></affiliation></author><author><name sortKey="Yogavel, Manickam" sort="Yogavel, Manickam" uniqKey="Yogavel M" first="Manickam" last="Yogavel">Manickam Yogavel</name><affiliation><nlm:aff id="N0x1ca9390N0x43159b8">Structural and Computational Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Road, New Delhi 110067, India, the</nlm:aff></affiliation></author><author><name sortKey="Kumar, Anuj" sort="Kumar, Anuj" uniqKey="Kumar A" first="Anuj" last="Kumar">Anuj Kumar</name><affiliation><nlm:aff id="N0x1ca9390N0x43159b8">Structural and Computational Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Road, New Delhi 110067, India, the</nlm:aff></affiliation></author><author><name sortKey="Belrhali, Hassan" sort="Belrhali, Hassan" uniqKey="Belrhali H" first="Hassan" last="Belrhali">Hassan Belrhali</name><affiliation><nlm:aff id="N0x1ca9390N0x43159b8">European Molecular Biology Laboratory, 6 Rue Jules Horowitz, BP 181, F-38042 Grenoble Cédex 9, France, the</nlm:aff></affiliation></author><author><name sortKey="Jain, S K" sort="Jain, S K" uniqKey="Jain S" first="S. K." last="Jain">S. K. Jain</name><affiliation><nlm:aff id="N0x1ca9390N0x43159b8">Department of Biotechnology, Hamdard University, Hamdard Nagar, New Delhi 110062, India, and the</nlm:aff></affiliation></author><author><name sortKey="Rug, Melanie" sort="Rug, Melanie" uniqKey="Rug M" first="Melanie" last="Rug">Melanie Rug</name><affiliation><nlm:aff id="N0x1ca9390N0x43159b8">Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia</nlm:aff></affiliation></author><author><name sortKey="Brown, Monica" sort="Brown, Monica" uniqKey="Brown M" first="Monica" last="Brown">Monica Brown</name><affiliation><nlm:aff id="N0x1ca9390N0x43159b8">Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia</nlm:aff></affiliation></author><author><name sortKey="Maier, Alexander G" sort="Maier, Alexander G" uniqKey="Maier A" first="Alexander G." last="Maier">Alexander G. Maier</name><affiliation><nlm:aff id="N0x1ca9390N0x43159b8">Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia</nlm:aff></affiliation></author><author><name sortKey="Sharma, Amit" sort="Sharma, Amit" uniqKey="Sharma A" first="Amit" last="Sharma">Amit Sharma</name><affiliation><nlm:aff id="N0x1ca9390N0x43159b8">Structural and Computational Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Road, New Delhi 110067, India, the</nlm:aff></affiliation></author></analytic><series><title level="j">The Journal of Biological Chemistry</title><idno type="ISSN">0021-9258</idno><idno type="eISSN">1083-351X</idno><imprint><date when="2009">2009</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Nucleosome assembly proteins (NAPs) are histone chaperones that are
essential for the transfer and incorporation of histones into nucleosomes.
NAPs participate in assembly and disassembly of nucleosomes and in chromatin
structure organization. Human malaria parasite <italic>Plasmodium falciparum</italic>contains two nucleosome assembly proteins termed PfNapL and PfNapS. To gain
structural insights into the mechanism of NAPs, we have determined and
analyzed the crystal structure of PfNapL at 2.3 Å resolution. PfNapL, an
ortholog of eukaryotic NAPs, is dimeric in nature and adopts a characteristic
fold seen previously for yeast NAP-1 and Vps75 and for human SET/TAF-1b
(β)/INHAT. The PfNapL monomer is comprised of domain I, containing a
dimerization α-helix, and a domain II, composed of α-helices and a
β-subdomain. Structural comparisons reveal that the “accessory
domain,” which is inserted between the domain I and domain II in yeast
NAP-1 and other eukaryotic NAPs, is surprisingly absent in PfNapL. Expression
of green fluorescent protein-tagged PfNapL confirmed its exclusive
localization to the parasite cytoplasm. Attempts to disrupt the PfNapL gene
were not successful, indicating its essential role for the malaria parasite. A
detailed analysis of PfNapL structure suggests unique histone binding
properties. The crucial structural differences observed between parasite and
yeast NAPs shed light on possible new modes of histone recognition by
nucleosome assembly proteins.</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></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">J Biol Chem</journal-id><journal-id journal-id-type="publisher-id">jbc</journal-id><journal-title>The Journal of Biological Chemistry</journal-title><issn pub-type="ppub">0021-9258</issn><issn pub-type="epub">1083-351X</issn><publisher><publisher-name>American Society for Biochemistry and Molecular Biology</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">19176479</article-id><article-id pub-id-type="pmc">2665062</article-id><article-id pub-id-type="publisher-id">10076</article-id><article-id pub-id-type="doi">10.1074/jbc.M808633200</article-id><article-categories><subj-group subj-group-type="heading"><subject>DNA: Replication, Repair, Recombination, and Chromosome Dynamics</subject></subj-group></article-categories><title-group><article-title>Crystal Structure of Malaria Parasite Nucleosome Assembly
Protein</article-title><subtitle><italic>DISTINCT MODES OF PROTEIN LOCALIZATION AND HISTONE
RECOGNITION</italic><xref ref-type="fn" rid="fn1">*</xref><xref ref-type="fn" rid="fn3">S⃞</xref></subtitle></title-group><contrib-group><contrib contrib-type="author"><name><surname>Gill</surname><given-names>Jasmita</given-names></name><xref ref-type="aff" rid="N0x1ca9390N0x43159b8">‡</xref><xref ref-type="fn" rid="fn4">1</xref></contrib><contrib contrib-type="author"><name><surname>Yogavel</surname><given-names>Manickam</given-names></name><xref ref-type="aff" rid="N0x1ca9390N0x43159b8">‡</xref><xref ref-type="fn" rid="fn4">1</xref></contrib><contrib contrib-type="author"><name><surname>Kumar</surname><given-names>Anuj</given-names></name><xref ref-type="aff" rid="N0x1ca9390N0x43159b8">‡</xref><xref ref-type="fn" rid="fn5">2</xref></contrib><contrib contrib-type="author"><name><surname>Belrhali</surname><given-names>Hassan</given-names></name><xref ref-type="aff" rid="N0x1ca9390N0x43159b8">§</xref></contrib><contrib contrib-type="author"><name><surname>Jain</surname><given-names>S. K.</given-names></name><xref ref-type="aff" rid="N0x1ca9390N0x43159b8">¶</xref></contrib><contrib contrib-type="author"><name><surname>Rug</surname><given-names>Melanie</given-names></name><xref ref-type="aff" rid="N0x1ca9390N0x43159b8">∥</xref></contrib><contrib contrib-type="author"><name><surname>Brown</surname><given-names>Monica</given-names></name><xref ref-type="aff" rid="N0x1ca9390N0x43159b8">∥</xref></contrib><contrib contrib-type="author"><name><surname>Maier</surname><given-names>Alexander G.</given-names></name><xref ref-type="aff" rid="N0x1ca9390N0x43159b8">∥</xref><xref ref-type="fn" rid="fn6">3</xref></contrib><contrib contrib-type="author"><name><surname>Sharma</surname><given-names>Amit</given-names></name><xref ref-type="aff" rid="N0x1ca9390N0x43159b8">‡</xref><xref ref-type="corresp" rid="cor1">4</xref></contrib></contrib-group><aff id="N0x1ca9390N0x43159b8"><label>‡</label>Structural and Computational Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Road, New Delhi 110067, India, the<label>§</label>European Molecular Biology Laboratory, 6 Rue Jules Horowitz, BP 181, F-38042 Grenoble Cédex 9, France, the<label>¶</label>Department of Biotechnology, Hamdard University, Hamdard Nagar, New Delhi 110062, India, and the<label>∥</label>Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia</aff><author-notes><fn id="fn4"><label>1</label><p>Supported by the <grant-sponsor>Wellcome
Trust</grant-sponsor>.</p></fn><fn id="fn5"><label>2</label><p>Supported by a <grant-sponsor>Department of Biotechnology, Government of
India</grant-sponsor>, fellowship.</p></fn><fn id="fn6"><label>3</label><p>An Australian Research Council Research Fellow.</p></fn><fn id="cor1"><label>4</label><p>An International Wellcome Trust Senior Research Fellow in Biomedical Sciences.
To whom correspondence should be addressed. Tel./Fax: 911126741731; E-mail:
<email>amit.icgeb@gmail.com</email>.
</p></fn></author-notes><pub-date pub-type="ppub"><day>10</day><month>4</month><year>2009</year></pub-date><pub-date pub-type="pmc-release"><day>10</day><month>4</month><year>2009</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on
/art/copyright. </pmc-comment>
<volume>284</volume><issue>15</issue><fpage>10076</fpage><lpage>10087</lpage><history><date date-type="received"><day>13</day><month>11</month><year>2008</year></date><date date-type="rev-recd"><day>9</day><month>1</month><year>2009</year></date></history><permissions><copyright-statement>Copyright © 2009, The American Society for Biochemistry and
Molecular Biology, Inc.</copyright-statement><license license-type="open-access"><p><italic>Author's Choice</italic></p><p><ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by-nc/3.0/">Creative
Commons Attribution Non-Commercial License</ext-link> applies to Author
Choice Articles</p></license></permissions><self-uri xlink:title="pdf" xlink:href="zbc01509010076.pdf"></self-uri><abstract><p>Nucleosome assembly proteins (NAPs) are histone chaperones that are
essential for the transfer and incorporation of histones into nucleosomes.
NAPs participate in assembly and disassembly of nucleosomes and in chromatin
structure organization. Human malaria parasite <italic>Plasmodium falciparum</italic>contains two nucleosome assembly proteins termed PfNapL and PfNapS. To gain
structural insights into the mechanism of NAPs, we have determined and
analyzed the crystal structure of PfNapL at 2.3 Å resolution. PfNapL, an
ortholog of eukaryotic NAPs, is dimeric in nature and adopts a characteristic
fold seen previously for yeast NAP-1 and Vps75 and for human SET/TAF-1b
(β)/INHAT. The PfNapL monomer is comprised of domain I, containing a
dimerization α-helix, and a domain II, composed of α-helices and a
β-subdomain. Structural comparisons reveal that the “accessory
domain,” which is inserted between the domain I and domain II in yeast
NAP-1 and other eukaryotic NAPs, is surprisingly absent in PfNapL. Expression
of green fluorescent protein-tagged PfNapL confirmed its exclusive
localization to the parasite cytoplasm. Attempts to disrupt the PfNapL gene
were not successful, indicating its essential role for the malaria parasite. A
detailed analysis of PfNapL structure suggests unique histone binding
properties. The crucial structural differences observed between parasite and
yeast NAPs shed light on possible new modes of histone recognition by
nucleosome assembly proteins.</p></abstract></article-meta><notes><fn-group><fn><p><italic>The atomic coordinates and structure factors (code 3FS3) have been
deposited in the Protein Data Bank, Research Collaboratory for Structural
Bioinformatics, Rutgers University, New Brunswick, NJ
(<ext-link ext-link-type="uri" xlink:href="http://www.rcsb.org/">http://www.rcsb.org/</ext-link>).</italic></p></fn><fn id="fn1"><label>*</label><p>This work was supported in part by a grant from the
<grant-sponsor>Department of Biotechnology, Govt. of India</grant-sponsor>.
The x-ray facility at the <grant-sponsor>International Centre for Genetic
Engineering and Biotechnology, New Delhi</grant-sponsor>, is funded by the
<grant-sponsor>Wellcome Trust</grant-sponsor>. The costs
of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked
“<italic>advertisement</italic>” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.</p></fn><fn id="fn3"><label></label><p>The on-line version of this article (available at
<ext-link ext-link-type="uri" xlink:href="http://www.jbc.org">http://www.jbc.org</ext-link>)
contains supplemental Figs. S1-S6 and Table S1.</p></fn><fn id="fn2"><label></label><p><italic>Author's Choice</italic>—Final version full
access.</p></fn></fn-group></notes></front><body><p>Chromatin assembly and remodeling are vital cellular processes that are
pivotal during replication, transcription, recombination, and repair in
eukaryotic cells. Nucleosomes are the fundamental repeating subunits of all
eukaryotic chromatin (except when packaged in sperm). Nucleosomes are made up
of DNA and four pairs of histone proteins, which together resemble
“beads on a string” when observed with an electron microscope.
Histones H2A, H2B, H3, and H4 are part of the nucleosome, whereas histone H1
sits on top of the structure, keeping in place the DNA that is wrapped around
the other histones (<xref ref-type="bibr" rid="ref1">1</xref>).
Nucleosome assembly is a fundamental biological process that is required for
the replication and maintenance of chromatin in the eukaryotic nucleus. In
dividing cells, newly synthesized DNA is assembled rapidly into chromatin by a
process that appears to involve an indirect coupling between DNA replication
and nucleosome assembly. The assembly of nucleosome occurs in a two-step
process: a tetramer of H3 and H4 is first deposited onto DNA, and then the
nucleosome core is completed by the addition of two heterodimers of H2A and
H2B (<xref ref-type="bibr" rid="ref1">1</xref>,
<xref ref-type="bibr" rid="ref2">2</xref>). Histones are highly basic
proteins and require chaperones that promote their proper interaction to form
chromatin. Histone chaperones also prevent nonspecific aggregation of DNA with
histones. A number of histone chaperones (<italic>e.g.</italic> nucleosome assembly
protein 1 (NAP-1),<xref ref-type="fn" rid="fn7">5</xref>nucleoplasmin, Asf1 (anti-silencing function), CAF-1 (chromatin assembly
factor), and HirA (histone regulator A)) are involved in nucleosome/chromatin
assembly/disassembly (<xref ref-type="bibr" rid="ref3">3</xref>).</p><p>Some histone chaperones, such as nucleoplasmin and NAP-1, exhibit a
preference for binding to histones H2A and H2B relative to histones H3 and H4
(<xref ref-type="bibr" rid="ref4">4</xref>,
<xref ref-type="bibr" rid="ref5">5</xref>). Other histone chaperones,
which include CAF-1, N1/N2, and Spt6, associate preferentially with H3 and H4.
Interestingly, it has also been observed that newly synthesized histones are
acetylated (such as at positions 5, 8, and 12 of histone H4) and then
subsequently deacetylated after assembly into chromatin
(<xref ref-type="bibr" rid="ref6">6</xref>,
<xref ref-type="bibr" rid="ref7">7</xref>). Thus, factors that mediate
histone acetylation or deacetylation may participate, perhaps indirectly by
the covalent modification of histones, in the chromatin assembly process.
Homologs of yeast NAP-1 are conserved among all eukaryotes, and yeast NAP-1 is
well characterized for its role in chromatin assembly
(<xref ref-type="bibr" rid="ref3">3</xref>,
<xref ref-type="bibr" rid="ref8">8</xref>). Gene knock-out phenotypes
in several studied organisms indicate essentiality of the nucleosome assembly
protein family; gene ablations in mouse and <italic>Drosophila</italic> cause
embryonic lethality, whereas in yeast, cells exhibit growth defects
(<xref ref-type="bibr" rid="ref9">9</xref>-<xref ref-type="bibr" rid="ref11">11</xref>).</p><p>Malaria is one of the most common infectious diseases and remains an
enormous public health problem. Malaria is caused by protozoan parasites of
the genus <italic>Plasmodium</italic>, and the most serious forms of the disease are
caused by <italic>Plasmodium falciparum</italic>(<xref ref-type="bibr" rid="ref12">12</xref>). It is therefore crucial
to identify, dissect, and exploit the molecular motors of malaria parasites,
which can serve as essential targets for antimalarials. The human malaria
parasite <italic>P. falciparum</italic> contains two nucleosome assembly proteins,
which we have termed PfNapL and PfNapS and which are orthologs of eukaryotic
NAPs (<xref ref-type="bibr" rid="ref13">13</xref>). We have shown that
both PfNapL and PfNapS are present in all erythrocytic stages of the parasite
(<xref ref-type="bibr" rid="ref13">13</xref>). PfNapL forms complexes
with both histone tetramer and octamer and is predominantly localized in the
cytoplasm in the asexual and sexual stages of the parasite
(<xref ref-type="bibr" rid="ref13">13</xref>,
<xref ref-type="bibr" rid="ref14">14</xref>). PfNapL by itself is
unable to deposit the histones onto DNA, but it can interact with both core
and linker histones and is involved in histone binding, shuttling, and
transfer/release, as shown earlier
(<xref ref-type="bibr" rid="ref13">13</xref>,
<xref ref-type="bibr" rid="ref14">14</xref>)
(<xref rid="fig1" ref-type="fig">Fig. 1<italic>f</italic></xref> reproduced
here from Ref. <xref ref-type="bibr" rid="ref14">14</xref>). The
histone binding characteristics of PfNapL have been detailed previously,
including its ability to transfer cytoplasmic histones on to PfNapS (which is
localized both in cytoplasm and the nucleus and transfers histones into the
nucleus for deposition) (<xref ref-type="bibr" rid="ref14">14</xref>).
A model for relay of histones from parasite cytoplasm to the nucleus has also
been proposed by us previously (see Ref.
<xref ref-type="bibr" rid="ref14">14</xref> and specifically
<xref rid="fig1" ref-type="fig">Fig. 1<italic>f</italic></xref>). PfNapL
preferentially interacts with the H3-H4 tetramer histones over H2A and H2B
histones. PfNapL and PfNapS do not interact with each other
(<xref ref-type="bibr" rid="ref14">14</xref>). To address the
structural basis of the nucleosome assembly activity in <italic>P.
falciparum</italic>, we have determined and analyzed the crystal structure of
PfNapL. Here, we detail the PfNapL structure and compare it with histone
chaperones like yeast NAP-1 (yNAP-1), human SET/TAF-1b (β)/INHAT (hSET),
Vps75, and Asf1-histone complex
(<xref ref-type="bibr" rid="ref15">15</xref>-<xref ref-type="bibr" rid="ref21">21</xref>)
in order to provide new insights into the mechanism of histone
recognition.</p><sec sec-type="methods"><title>EXPERIMENTAL PROCEDURES</title><p><italic>Protein Crystallization and Data Collection</italic>—PfNapL crystals
were obtained at 20 °C by the hanging drop vapor diffusion method using l
μl of full-length purified PfNapL protein
(<xref ref-type="bibr" rid="ref13">13</xref>,
<xref ref-type="bibr" rid="ref14">14</xref>) (3 mg ml<sup>-1</sup> in
buffer containing 25 m<sc>m</sc> Tris and 25 m<sc>m</sc> NaCl) and l μl
of 0.2 <sc>m</sc> MgCl<sub>2</sub> with 20% polyethylene glycol 3350 (mother
liquor). A single crystal was transferred to cryoprotectant containing a
higher concentration of mother liquor (30% polyethylene glycol 3350 and 0.3
<sc>m</sc> MgCl<sub>2</sub>) supplemented with 0.5 <sc>m</sc> NaI for 30 s
and flash-frozen under a stream of nitrogen gas at 100 K. X-ray diffraction
data were collected on an in-house rotating anode MICRO MAX 007 x-ray
generator (wavelength of 1.54 Å; Rigaku/MSC) operated at 40 kV and 20 mA
with Osmic mirrors (Vari Max HR). The images were recorded using an MAR345dtb
imaging plate, and the iodide-single anomalous dispersion (SAD) data set was
collected to 3.0 Å resolution. The iodide-soaked crystals of PfNapL
belong to the monoclinic space group C2 with cell dimensions of <italic>a</italic> =
76.68 Å, <italic>b</italic> = 37.83 Å, <italic>c</italic> = 79.31 Å, and
β = 99.83°, having one monomer per asymmetric unit. High resolution
native diffraction data to 2.3 Å resolution were collected at the BM14
beamline (European Synchotron Radiation Facility, Grenoble, France). These
crystals also belong to the monoclinic space group C2 with different cell
dimensions of <italic>a</italic> = 92.41 Å, <italic>b</italic> = 38.12 Å,
<italic>c</italic> = 80.05 Å, and β = 107.87°. The diffraction images
were processed and scaled with the HKL2000 suite
(<xref ref-type="bibr" rid="ref22">22</xref>).</p><p><italic>Phasing, Structure Determination, Refinement, and
Analysis</italic>—The structure was determined using the iodide-SAD
technique, and phasing was achieved by utilizing four iodide sites to 3.0
Å resolution using PHENIX
(<xref ref-type="bibr" rid="ref23">23</xref>). An initial model was
built automatically without side chains and subsequently rebuilt manually
using COOT (<xref ref-type="bibr" rid="ref24">24</xref>). The high
resolution structure was determined by molecular replacement technique using
the PfNapL iodide-SAD model. This model to 2.3 Å resolution was further
refined using CNS (<xref ref-type="bibr" rid="ref25">25</xref>)
(<xref ref-type="table" rid="tbl1">Table 1</xref>). The final model was
validated using PROCHECK (<xref ref-type="bibr" rid="ref26">26</xref>).
All figures were generated using Chimera
(<xref ref-type="bibr" rid="ref27">27</xref>). The least square
fittings and structural alignment were carried out using LSQMAN
(<xref ref-type="bibr" rid="ref28">28</xref>). Electrostatic potential
surfaces were generated using GRASP
(<xref ref-type="bibr" rid="ref29">29</xref>).</p><p><table-wrap position="float" id="tbl1"><label>TABLE 1</label><caption><p><bold>Data collection and refinement statistics</bold></p></caption><table frame="hsides" rules="groups"><thead><tr><th colspan="1" rowspan="1" align="center" valign="bottom"><bold>Parameter</bold></th><th colspan="1" rowspan="1" align="center" valign="bottom"><bold>Native</bold></th><th colspan="1" rowspan="1" align="center" valign="bottom"><bold>Iodide-SAD</bold></th></tr></thead><tbody><tr><td colspan="1" rowspan="1" align="left" valign="bottom"><bold>Data collection</bold></td><td colspan="1" rowspan="1" align="center" valign="bottom"></td><td colspan="1" rowspan="1" align="center" valign="bottom"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="bottom"> Cell dimensions
</td><td colspan="1" rowspan="1" align="center" valign="bottom"><italic>a</italic> = 92.41 Å, <italic>b</italic> = 38.12 Å, <italic>c</italic> = 80.05
Å, and β = 107.87°
</td><td colspan="1" rowspan="1" align="center" valign="bottom"><italic>a</italic> = 76.68 Å, <italic>b</italic> = 37.83 Å, <italic>c</italic> = 79.31
Å, and β = 99.83°
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="bottom"> Wavelength (Å)
</td><td colspan="1" rowspan="1" align="center" valign="bottom"> 1.0
</td><td colspan="1" rowspan="1" align="center" valign="bottom"> 1.54
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="bottom"> Anomalous scatterer
</td><td colspan="1" rowspan="1" align="center" valign="bottom"></td><td colspan="1" rowspan="1" align="center" valign="bottom"> I
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="bottom"> Resolution (Å)
</td><td colspan="1" rowspan="1" align="center" valign="bottom"> 50-2.3
</td><td colspan="1" rowspan="1" align="center" valign="bottom"> 50-3.0
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="bottom"> Outer shell resolution
(Å)<xref ref-type="table-fn" rid="tblfn1"><italic>a</italic></xref></td><td colspan="1" rowspan="1" align="center" valign="bottom"> 2.38-2.3
</td><td colspan="1" rowspan="1" align="center" valign="bottom"> 3.11-3.0
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="bottom"> No. of unique reflections
</td><td colspan="1" rowspan="1" align="center" valign="bottom"> 12,065 (1217)
</td><td colspan="1" rowspan="1" align="center" valign="bottom"> 4606 (451)
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="bottom"> Redundancy
</td><td colspan="1" rowspan="1" align="center" valign="bottom"> 5.1 (5.1)
</td><td colspan="1" rowspan="1" align="center" valign="bottom"> 14.5 (13.2)
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="bottom"><italic>R</italic><sub>merge</sub><xref ref-type="table-fn" rid="tblfn2"><italic>b</italic></xref></td><td colspan="1" rowspan="1" align="center" valign="bottom"> 0.052 (0.238)
</td><td colspan="1" rowspan="1" align="center" valign="bottom"> 0.084 (0.331)
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="bottom"> Completeness (%)
</td><td colspan="1" rowspan="1" align="center" valign="bottom"> 100 (99.9)
</td><td colspan="1" rowspan="1" align="center" valign="bottom"> 99.0 (97.6)
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="bottom"><italic>I</italic>/σ<italic>I</italic></td><td colspan="1" rowspan="1" align="center" valign="bottom">26.1 (4.98)
</td><td colspan="1" rowspan="1" align="center" valign="bottom">45.6 (11.4)
</td></tr></tbody><tbody><tr><td colspan="1" rowspan="1" align="left" valign="bottom"><bold>Phasing (PHENIX)</bold></td><td colspan="1" rowspan="1" align="center" valign="bottom"></td><td colspan="1" rowspan="1" align="center" valign="bottom"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="bottom"> AutoSol figure of merit
</td><td colspan="1" rowspan="1" align="center" valign="bottom"></td><td colspan="1" rowspan="1" align="center" valign="bottom"> 0.36
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="bottom">Post DM-figure of merit
</td><td colspan="1" rowspan="1" align="center" valign="bottom"></td><td colspan="1" rowspan="1" align="center" valign="bottom">0.66
</td></tr></tbody><tbody><tr><td colspan="1" rowspan="1" align="left" valign="bottom"><bold>Refinement</bold></td><td colspan="1" rowspan="1" align="center" valign="bottom"></td><td colspan="1" rowspan="1" align="center" valign="bottom"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="bottom"> Resolution range (Å)
</td><td colspan="1" rowspan="1" align="center" valign="bottom"> 50-2.3
</td><td colspan="1" rowspan="1" align="center" valign="bottom"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="bottom"> No. of reflections (test set)
</td><td colspan="1" rowspan="1" align="center" valign="bottom"> 1219
</td><td colspan="1" rowspan="1" align="center" valign="bottom"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="bottom"><italic>R</italic><sub>factor</sub>/<italic>R</italic><sub>free</sub> (%)
</td><td colspan="1" rowspan="1" align="center" valign="bottom"> 21.8/28.5
</td><td colspan="1" rowspan="1" align="center" valign="bottom"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="bottom"> No. of protein atoms
</td><td colspan="1" rowspan="1" align="center" valign="bottom"> 1668
</td><td colspan="1" rowspan="1" align="center" valign="bottom"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="bottom">No. of water molecules
</td><td colspan="1" rowspan="1" align="center" valign="bottom">74
</td><td colspan="1" rowspan="1" align="center" valign="bottom"></td></tr></tbody><tbody><tr><td colspan="1" rowspan="1" align="left" valign="bottom"><bold><italic>B</italic> factor (Å<sup>2</sup>)</bold></td><td colspan="1" rowspan="1" align="center" valign="bottom"></td><td colspan="1" rowspan="1" align="center" valign="bottom"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="bottom"> Protein atoms
</td><td colspan="1" rowspan="1" align="center" valign="bottom"> 36.6
</td><td colspan="1" rowspan="1" align="center" valign="bottom"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="bottom">Water atoms
</td><td colspan="1" rowspan="1" align="center" valign="bottom">35.1
</td><td colspan="1" rowspan="1" align="center" valign="bottom"></td></tr></tbody><tbody><tr><td colspan="1" rowspan="1" align="left" valign="bottom"><bold>Stereochemistry</bold></td><td colspan="1" rowspan="1" align="center" valign="bottom"></td><td colspan="1" rowspan="1" align="center" valign="bottom"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="bottom"> r.m.s. deviation bond length (Å)
</td><td colspan="1" rowspan="1" align="center" valign="bottom"> 0.01
</td><td colspan="1" rowspan="1" align="center" valign="bottom"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="bottom">r.m.s. deviation bond angle (degrees)
</td><td colspan="1" rowspan="1" align="center" valign="bottom">1.50
</td><td colspan="1" rowspan="1" align="center" valign="bottom"></td></tr></tbody><tbody><tr><td colspan="1" rowspan="1" align="left" valign="bottom"><bold>Ramachandran plot</bold></td><td colspan="1" rowspan="1" align="center" valign="bottom"></td><td colspan="1" rowspan="1" align="center" valign="bottom"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="bottom"> In most favored regions (%)
</td><td colspan="1" rowspan="1" align="center" valign="bottom"> 91.5
</td><td colspan="1" rowspan="1" align="center" valign="bottom"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="bottom"> In additionally allowed regions (%)
</td><td colspan="1" rowspan="1" align="center" valign="bottom"> 7.4
</td><td colspan="1" rowspan="1" align="center" valign="bottom"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="bottom">In generously allowed regions (%)
</td><td colspan="1" rowspan="1" align="center" valign="bottom">1.1
</td><td colspan="1" rowspan="1" align="center" valign="bottom"></td></tr></tbody></table><table-wrap-foot><fn id="tblfn1"><label>a</label><p>Numbers in parentheses are values in the highest resolution shell.</p></fn><fn id="tblfn2"><label>b</label><p><italic>R</italic><sub>merge</sub> = ∑|<italic>I</italic><sub>obs</sub> -
<<italic>I</italic>>|/∑<<italic>I</italic>> summed over all
observations and reflections.</p></fn></table-wrap-foot></table-wrap></p><p><italic>Parasite Culture, Plasmid Constructs, and
Transfection</italic>—<italic>P. falciparum</italic> of the 3D7 strain were cultured
<italic>in vitro</italic> in O+ human erythrocytes using standard conditions
(<xref ref-type="bibr" rid="ref30">30</xref>). For the gene deletion
construct, we amplified the 5′- and 3′-targeting segments of
PfNapL using the primer pairs aw775/aw776 and aw777/778, resulting in 645- and
622-bp products, respectively. After amplification, the targeting segments
were cloned into the pCC-1 vector
(<xref ref-type="bibr" rid="ref31">31</xref>) using SacII/SpeI for the
5′ and EcoRI/AvrII for the 3′ segment, resulting in the
pCC-1ΔPfNapL plasmid. After proliferation of the plasmid DNA in
<italic>Escherichia coli</italic> PMC103 cells and purification with a Qiagen Maxi
kit, 80 μg of plasmid DNA was taken up in cytomix and transfected via
electroporation using standard procedures
(<xref ref-type="bibr" rid="ref32">32</xref>). Parasites containing the
plasmid were selected for in the presence of 2 n<sc>m</sc> WR99210
(<xref ref-type="bibr" rid="ref33">33</xref>). The resulting parasite
population was then subjected to two off/on drug cycles for 21 days each to
encourage the loss of free plasmid. After each addition of WR99210, a
subpopulation was selected on 231 n<sc>m</sc> 5-fluorocytosine in the
presence of WR99210 (to encourage double recombination events) and analyzed
via Southern blot. Two independent rounds of transfections were performed and
analyzed: Aw775, atcccgcggTATTTGAATTTGATTTCTTGC; Aw776,
gatactagtGCTAGTTAAATAAACATAACAG; Aw777, atcgaattcGTATGCAACTACTTTTTCG; Aw778,
gatcctaggCTGTATGTAATAACACTGGCTCG.</p><p>To generate transgenic parasites expressing GFP chimeras, we amplified the
sequence encoding PfNapL from cDNA and cloned the product into the pARL vector
(<xref ref-type="bibr" rid="ref34">34</xref>) to create PfNapL-pARL.
Conserved residues of the putative nuclear export signal (NES) were mutated by
yielding the plasmids PfNapL L46A-pARL and PfNapL Q50A-pARL. The expression of
chimeric proteins in this vector is driven by the <italic>crt</italic> promoter. The
correctness of each construct was confirmed by DNA sequencing. 3D7 <italic>P.
falciparum</italic> parasites were transfected with 100 μg of plasmid DNA
(Qiagen) by electroporation and cultured in the presence of 2 n<sc>m</sc>WR99210 (<xref ref-type="bibr" rid="ref32">32</xref>).</p><p><italic>Microscopy</italic>—In order to observe the localization of the GFP
chimeras in all three generated cell lines, live cells were stained with DAPI
and immediately processed for viewing at ambient temperature. Cells were
viewed with an Apochromat ×100/1.4 numerical aperture oil DIC lens on a
Zeiss Axioskop 2 microscope equipped with a PCO SensiCam (12-bit) camera and
Axiovision 3 software. Captured images were processed using Photoshop and
ImageJ software (available on the World Wide Web).</p><p><italic>Histone-binding ELISAs</italic>—Residues Asp<sup>192</sup>,
His<sup>227</sup>, Thr<sup>230</sup>, Glu<sup>260</sup>, Lys<sup>266</sup>,
Asp<sup>233</sup>, Tyr<sup>259</sup>, Ile<sup>136</sup>, and Ile<sup>147</sup>were mutated to alanine using the QuikChange site-directed mutagenesis kit
(Stratagene). Tetramer and octamer forms of calf thymus histone were
reconstituted from individual histones (Roche Applied Science) after
denaturing and refolding according to the method developed by Tanaka <italic>et
al.</italic> (<xref ref-type="bibr" rid="ref35">35</xref>). ELISAs were
performed by coating individual histones, histone tetramers, or histone
octamers (100 ng/well) in microtiter plates (Nunc). The histones were coated
on plates in phosphate-buffered saline (PBS) and incubated at 4 °C
overnight. The following day, the plates were washed with PBS plus 0.2% Tween
20 (PBST) and blocked with 3% PBS-bovine serum albumin (200 μl/well) and
incubated for 1 h at 37 °C. Then 100 μl of purified PfNapL
(concentration ranging from 25 to 800 ng) was added to each well in
histone-binding buffer (20 m<sc>m</sc> HEPES, 7.5 m<sc>m</sc>MgCl<sub>2</sub>, 1 m<sc>m</sc> dithiothreitol, 0.5 m<sc>m</sc> EDTA, and
50 m<sc>m</sc> KCl, pH 7.5). Plates were incubated for an additional 1 h at
37 °C and washed three times with PBST, and then PfNapL anti-rabbit
polyclonal antibodies were added to each well (1:25,000 dilutions). After
incubation at 37 °C for 1 h, three more PBST washes were done, followed by
the addition of horseradish peroxidase-conjugated secondary antibody
(anti-rabbit), and incubated for an additional 1 h at 37 °C. After three
more PBST washes, a color reaction was developed using orthophenylenediamine
(Sigma) and H<sub>2</sub>O<sub>2</sub> in citrate-phosphate buffer. The
optical density was measured at 490 nm using an ELISA plate reader (Molecular
Probes). Bovine serum albumin, primary antibodies, and secondary antibodies
were separately used as controls in these ELISAs.</p></sec><sec><title>RESULTS</title><p><italic>Structure Determination and Overall Structure of PfNapL</italic>—The
crystal structure of PfNapL was determined using the iodide-SAD technique at
3.0 Å and refined using high resolution data to 2.3 Å resolution
(<xref ref-type="table" rid="tbl1">Table 1</xref>). Purified PfNapL
crystallized in monoclinic space group C2 with solvent content of 55%, and the
asymmetric unit contains one monomer of PfNapL. For a total of 347 residues of
full-length PfNapL (molecular mass ∼41 kDa), electron density was observed
for the central core residues 33-281. The N- and C-terminal regions in PfNapL
were presumably cleaved off during protein processing prior to
crystallization. The regions 89-91, 138-144, 171-178, 200-221, and 235-246
have weak electron density and are disordered in the overall structure. The
final refined model of PfNapL has <italic>R</italic><sub>factor</sub> and
<italic>R</italic><sub>free</sub> values of 21.8 and 28.5%, respectively
(<xref ref-type="table" rid="tbl1">Table 1</xref>).</p><p>The NAP/SET proteins consist of a central region of ∼250 residues that
are thought to be primarily responsible for histone binding
(<xref rid="fig1" ref-type="fig">Fig. 1<italic>a</italic></xref>). In general,
NAPs comprise low complexity sequences in their N and C termini, wherein the C
terminus usually contains stretches of acidic residues
(<xref rid="fig1" ref-type="fig">Fig. 1<italic>a</italic></xref>). The overall
fold of PfNapL is similar to yNAP-1 and hSET
(<xref ref-type="bibr" rid="ref15">15</xref>,
<xref ref-type="bibr" rid="ref16">16</xref>)
(<xref rid="fig1" ref-type="fig">Fig. 1<italic>b</italic></xref>). Each
monomer contains a domain I composed of a dimerization helix α2
(residues 37-87). The monomer also contains domain II, which is composed of
α-helices (α3 (residues 102-108), α4 (residues 111-116), and
α5 (residues 119-127)), a β subdomain containing four antiparallel
β-strands (residues 128-185), another α-helix α7 (residues
225-228), and a final two α-helices on the other side of the β
subdomain (α7 (residues 248-267) and α8 (residues 269-277))
(<xref rid="fig1" ref-type="fig">Fig. 1<italic>b</italic></xref>). We have
earlier shown the dimeric nature of PfNapL in solution
(<xref ref-type="bibr" rid="ref13">13</xref>). The crystallographic
2-fold symmetry generates a homodimer of PfNapL of ∼87 × 52 ×
33 Å with a total buried surface area of 4209 Å<sup>2</sup>. The
51-residue-long dimerization helix α2 forms the characteristic shape of
PfNapL, wherein two backbone helices cluster in an antiparallel manner to form
the dimer using mainly hydrophobic interactions and few salt bridges/hydrogen
bonds (<xref rid="fig1" ref-type="fig">Fig. 1<italic>b</italic></xref>). The
overall sequence identity of PfNapL with yNAP-1, hSET, and Vps75 is 24, 25,
and 14%, respectively.
(<xref ref-type="bibr" rid="ref15">15</xref>-<xref ref-type="bibr" rid="ref19">19</xref>)
(<xref rid="fig1" ref-type="fig">Fig. 1<italic>c</italic></xref> and Fig. S1).
The phylogenetic analysis of NAPs suggests greater evolutionary distance of
malaria parasite NAPs from homologs in yeast and humans (available on the
World Wide Web) (<xref rid="fig1" ref-type="fig">Fig.
1<italic>e</italic></xref>).</p><p><italic>Gene Knock-out Studies on PfNapL</italic>—To investigate whether the
function of PfNapL could be disrupted, we designed a construct to specifically
target the PfNapL gene and to replace it with an human dihydrofolate
reductase-selectable marker cassette. Despite several attempts, we were not
able to obtain a population where the PfNapL gene was replaced by the human
dihydrofolate reductase cassette (Fig. S2). Although we cannot exclude any
technical problems, the inability to disrupt the function of PfNapL is
indicative of a pivotal role of this gene product for the survival of <italic>P.
falciparum</italic>. At the moment, our repertoire of molecular tools in <italic>P.
falciparum</italic> precludes us from performing conditional gene disruptions. An
essential or at least a very beneficial role of this molecule has been
described in several other organisms like mouse and <italic>Drosophila</italic>(<xref ref-type="bibr" rid="ref9">9</xref>,
<xref ref-type="bibr" rid="ref10">10</xref>). Therefore, we propose
that the PfNapL protein is very likely to be essential for parasite survival
and that it provides a potential new target for disruption of parasite
nucleosome assembly as a route toward controlling parasite growth.</p><p><fig position="float" id="fig1"><label>FIGURE 1.</label><caption><p><bold>The overall structure of PfNapL.</bold> <italic>a</italic>, domain diagram for the
NAP/SET family of proteins. Histone chaperone proteins comprise a central
domain and low complexity N and C termini. The inserted accessory domain in
yNAP-1 (yellow) is highlighted, which is missing both in PfNapL and hSET (set
to scale). The <italic>brown bar</italic> is not set to scale, since the N and C
termini are variable. <italic>b</italic>, structure of the PfNapL dimer generated
using 2-fold crystallographic symmetry operation. Each monomer of PfNapL
contains a domain I composed of dimerization helix α2 and domain II
composed of a β subdomain (four antiparallel β strands) and
α-helices on the other side. <italic>c</italic>, structure-based sequence
alignment of PfNapL, yNAP-1, and hSET. The structures share high structural
homology, except for an additional accessory domain between domain I and
domain II in yNAP-1 (<italic>foreground colored yellow</italic>). Residues
contributing to dimer formation are <italic>colored green</italic>. Conserved
hydrophobic motifs, predicted NES, and predicted NLS in PfNapL are
<italic>underlined</italic> in <italic>purple</italic> and <italic>blue</italic>, respectively.
Conserved residues from the hSET mutagenesis data are <italic>colored red</italic> and
<italic>blue. d</italic>, sequence alignment of PfNapL and other eukaryotic members of
the NAP family: yNAP-1 (<italic>Saccharomyces cerevisiae</italic>), xNAP-1
(<italic>Xenopus laevis</italic>), dNAP-1 (<italic>Drosophila melanogaster</italic>), and
hNAP-1 (<italic>Homo sapiens</italic>). The accessory domain in yNAP-1 is
<italic>foreground-colored yellow</italic>. The identical/conserved surface residues
and conserved hydrophobic motifs are <italic>colored pink</italic> and
<italic>purple</italic>, respectively. <italic>e</italic>, phylogenetic tree of NAPs from
various species showing greater evolutionary distance of malaria parasite NAPs
from homologs in yeast and humans (indicated by <italic>red arrows</italic>).
<italic>f</italic>, model for <italic>in vitro</italic> relay of histones by <italic>P.
falciparum</italic> nucleosome assembly proteins. This model was reproduced from
Ref. <xref ref-type="bibr" rid="ref14">14</xref>, showing distinct
roles for PfNapL (in cytoplasm as a histone carrier) and PfNapS (in nucleus
capable of histone deposition). PfNapL (<italic>L</italic>) interacts with histones
and can deliver histones to PfNapS (<italic>S</italic>; phosphorylated or
unphosphorylated) readily. PfNapL upon phosphorylation
(<italic>L<sup>p</sup></italic>) binds 3-fold better to the histones when compared
with the unphosphorylated form of PfNapL. Phospho-PfNapL may deliver its
histone cargo PfNapS. The latter takes over histones from
Phospho-PfNapL-histone complexes and shuttles the histones into nucleus.</p></caption><graphic xlink:href="zbc017097122001a"></graphic><graphic xlink:href="zbc017097122001f"></graphic></fig></p><p><italic>Structural Conservation with yNAP-1, hSET, and Vps75</italic>—The
root mean square (r.m.s.) deviation derived from the least square fittings of
191 C<sup>α</sup> of PfNapL with the corresponding C<sup>α</sup>of yNAP-1, hSET, and Vps75 is 1.9, 1.8, and 1.8 Å, respectively
(<xref rid="fig2" ref-type="fig">Fig. 2<italic>a</italic></xref> and Fig. S3).
The dimerization helix α2 of PfNapL shows an r.m.s. deviation of 1.4 and
1.1 Å with the corresponding dimerization helices of yNAP-1 and hSET
(<xref rid="fig2" ref-type="fig">Fig. 2, <italic>b</italic> and
<italic>c</italic></xref>). The core domain II of PfNapL is also similar to
yNAP-1 and hSET, with an average r.m.s. deviation of 1.1 and 1.0 Å,
respectively (<xref rid="fig2" ref-type="fig">Fig. 2, <italic>b</italic> and
<italic>c</italic></xref>). The dimerization helix α2 of PfNapL has high
sequence similarity with the corresponding dimerization helices of hSET/yNAP-1
for a total of ∼50 residues (<xref rid="fig2" ref-type="fig">Fig. 2,
<italic>b</italic> and <italic>c</italic></xref>). In the β subdomain, PfNapL has 16
of 24 identical and 5 of 8 conserved residues with the corresponding β
subdomains of hSET/yNAP-1. The residues 290-295 of this antiparallel
β-sheet region of yNAP-1 had been previously identified as the nuclear
localization signal (NLS) (<xref ref-type="bibr" rid="ref15">15</xref>)
(Figs. <xref rid="fig1" ref-type="fig">1<italic>c</italic></xref> and
<xref rid="fig2" ref-type="fig">2, <italic>b</italic> and <italic>c</italic></xref>).
Residues 200-221 in PfNapL corresponding to this short anti-parallel
β-sheet region (β5 and β6) in yNAP-1 are disordered
(<xref rid="fig2" ref-type="fig">Fig. 2<italic>b</italic></xref>). However,
the NLS sequences between yeast and PfNapL are highly conserved (Figs.
<xref rid="fig1" ref-type="fig">1<italic>c</italic></xref> and
<xref rid="fig2" ref-type="fig">2<italic>b</italic></xref>).</p><p><italic>PfNapL Lacks the “Accessory Domain” Conserved in Higher
Eukaryotic NAPs</italic>—Comparison between yNAP-1, hSET, and PfNapL
structures revealed that yNAP-1 contains an additional α-helix, termed
the “accessory domain,” which has been inserted between domain I
and II but is absent in both hSET and PfNapL (Figs.
<xref rid="fig1" ref-type="fig">1<italic>c</italic></xref> and
<xref rid="fig2" ref-type="fig">2<italic>a</italic></xref>). Based on the
sequence comparison, it can be inferred that this accessory domain is present
in all other eukaryotic nucleosome assembly proteins but is seen to be
specifically absent in PfNapL and other NAPs from <italic>Plasmodium</italic> species
(<xref rid="fig1" ref-type="fig">Fig. 1<italic>d</italic></xref>).
Intriguingly, SET protein homologs, such as the recently characterized human
SET (<xref ref-type="bibr" rid="ref16">16</xref>), are absent in yeast
and <italic>Plasmodium</italic> (stand alone SET domains are found in higher
eukaryotes like mice, cows, and humans). In <italic>Plasmodium</italic>, SET-like
domains are a constituent of significantly larger proteins (molecular mass
≥142 kDa) termed “SET domain proteins,” where functions and
localizations are still unknown (PFD0190w, PFF1440w, and PF08_0012).</p><p><italic>PfNapL Dimer and Conserved Hydrophobic Motifs</italic>—The dimer
formation in yNAP-1, hSET, Vps75, and PfNapL is achieved via the dimerization
helix α2. In PfNapL, residues Gln<sup>50</sup>, Glu<sup>52</sup>,
Tyr<sup>54</sup>, Glu<sup>57</sup>, Lys<sup>59</sup>, Glu<sup>63</sup>,
Arg<sup>68</sup>, Lys<sup>70</sup>, Tyr<sup>71</sup>, and Lys<sup>81</sup> of
the dimerization helix α2 and Pro<sup>271</sup>, Tyr<sup>272</sup>, and
Asp<sup>275</sup> of helix α8, which lies on the other side of the
β subdomain contribute to dimer formation. Residues Glu<sup>52</sup> and
Glu<sup>63</sup> and Lys<sup>70</sup> and Lys<sup>81</sup> of the dimerization
helix α2 form salt bridges. The hydrogen bonding interactions
contributing to dimer formation in PfNapL are mostly structurally conserved
with those of hSET (<xref rid="fig1" ref-type="fig">Fig.
1<italic>c</italic></xref>). Unlike yNAP-1, the PfNapL dimerization helices are
bent at ∼50° very similar to the hSET and Vps75 dimers (Fig.
S4<italic>a</italic> and Fig. S5<italic>a</italic>). Although a proline residue at position 77
is present in PfNapL, there are no kinks formed in the dimerization helices
upon dimer formation in PfNapL in contrast to yNAP-1 (Fig. S4<italic>a</italic>).
Interestingly, PfNapL dimer displays a somewhat narrow cavity (Fig.
S5<italic>b</italic>) when compared with Vps75 dimer
(<xref ref-type="bibr" rid="ref18">18</xref>,
<xref ref-type="bibr" rid="ref19">19</xref>). In yNAP-1, a disulfide
bond is formed by residues Cys<sup>249</sup> and Cys<sup>272</sup>. These
positions are replaced by serine and threonine in PfNapL, and only a single
cysteine is present in PfNapL at position 133. The hydrophobic core of PfNapL
and yNAP-1 is stabilized by the presence of SFF(T/N)FF and (L/I)P(E/S)FWL
motifs that are conserved among the NAP family
(<xref rid="fig1" ref-type="fig">Fig. 1<italic>c</italic></xref> and Fig.
S4<italic>b</italic>).</p><p><fig position="float" id="fig2"><label>FIGURE 2.</label><caption><p><bold>Comparison of PfNapL structure with yNAP-1 (Protein Data Bank code 2AYU)
and hSET (Protein Data Bank code 2E50).</bold> <italic>a</italic>, superimposition of
PfNapL monomer (<italic>purple</italic>) onto yNAP-1 (<italic>blue</italic>) and hSET
(<italic>orange</italic>). Accessory domain of yNAP-1 is <italic>colored yellow</italic>. NES
and NLS in yNAP-1 and PfNapL are indicated. <italic>b</italic>, superimposition of
PfNapL and yNAP-1 dimers. The <italic>lower half</italic> shows superimposition of
domain I and II from PfNapL and yNAP-1 monomers. yNAP-1 is <italic>colored
blue</italic>, and the accessory domain is <italic>colored yellow</italic>. PfNapL is
<italic>colored purple</italic>. The extra residues in domain II of yNAP-1 are
<italic>colored cyan</italic> and are disordered in PfNapL. <italic>c</italic>,
superimposition of PfNapL and hSET dimers. The <italic>lower half</italic> shows
superimposition of domain I and II from PfNapL and hSET monomers. The extra
residues of PfNapL are <italic>colored cyan</italic> and are disordered in hSET.</p></caption><graphic xlink:href="zbc0170971220002"></graphic></fig></p><p><italic>PfNapL Localization in Parasite Cytoplasm Using Transfection
Studies</italic>—We have earlier shown that PfNapL is expressed during all
of the blood stages and is localized to the parasite cytoplasm
(<xref ref-type="bibr" rid="ref13">13</xref>,
<xref ref-type="bibr" rid="ref14">14</xref>). PfNapL also contains
conserved NLS and NES motifs (Figs.
<xref rid="fig1" ref-type="fig">1<italic>c</italic></xref> and
<xref rid="fig3" ref-type="fig">3, <italic>b</italic> and <italic>c</italic></xref>)
like the yNAP-1 (which is observed in both the cytoplasm and the nucleus).
Corresponding to the presence of NES in yNAP1 (residues 88-103)
(<xref rid="fig3" ref-type="fig">Fig. 3<italic>a</italic></xref>), residues
35-50 of PfNapL have been predicted as an NES
(<xref rid="fig3" ref-type="fig">Fig. 3, <italic>b</italic> and
<italic>c</italic></xref>). It has been suggested in yNAP-1 that this NES is
masked by the accessory domain, which is thought to play an important role in
the import and export of yNAP-1 into and from the nucleus
(<xref ref-type="bibr" rid="ref15">15</xref>)
(<xref rid="fig3" ref-type="fig">Fig. 3<italic>a</italic></xref>). As
mentioned earlier, this accessory domain in yNAP-1 (residues 141-180) is the
inserted α-helix, which is absent in PfNapL (Figs.
<xref rid="fig1" ref-type="fig">1<italic>c</italic></xref> and
<xref rid="fig2" ref-type="fig">2<italic>a</italic></xref>). Interestingly,
hSET also lacks this accessory domain yet contains both NES and NLS motifs
(<xref ref-type="bibr" rid="ref16">16</xref>). Further, hSET is
localized both in the cell cytoplasm and the nucleus, similar to yNAP-1
(<xref ref-type="bibr" rid="ref36">36</xref>) and like other histone
shuttling proteins. In this respect, PfNapL clearly stands as an exception
despite its structural and sequence similarities with yeast NAP and human SET
proteins.</p><p>In order to address whether PfNapL is located at some stage in the nucleus
and whether its cytosolic localization is due to continuous export mediated by
its putative NES, we generated cell lines expressing PfNapL-GFP chimeras. For
these chimeras, we either used wild-type sequence or introduced mutations in
the conserved residues of the NES (PfNapL L46A or PfNapL Q50A)
(<xref rid="fig3" ref-type="fig">Fig. 3<italic>c</italic></xref>). Disruption
of a functional NES was predicted to lead to the accumulation of GFP chimera
in the parasite nucleus. Expression of the GFP chimeras was verified via
Western blot and probed with anti-GFP antibodies
(<xref rid="fig4" ref-type="fig">Fig. 4<italic>a</italic></xref>). A band of
∼70 kDa was observed in all cell lines, corresponding to the size of
PfNapL (40.5 kDa) fused to GFP (26.8 kDa). The GFP fusion protein of all three
transfectants (wild type and mutants) was detectable in the cytoplasm of all
stages. Previous localization experiments with PfNapL-specific antibodies
detected an exclusive cytoplasmic localization in all stages, except for the
schizont stages, where the separation between nuclear (DAPI) and PfNapL
staining was less defined
(<xref ref-type="bibr" rid="ref13">13</xref>). This could be due to
greater interdigitation of the nucleus and cytoplasm at that stage or due to a
closer association of the PfNapL pool with the nucleus. To address this
question, we followed the localization of PfNapL wild type over the asexual
erythrocytic life cycle (<xref rid="fig4" ref-type="fig">Fig. 4,
<italic>b-d</italic></xref>). PfNapL wild type-GFP was detected in the cytoplasm
of the parasites. No fluorescence could be detected in the erythrocyte
cytosol. Neither the food vacuole nor the nucleus of the parasite showed
fluorescence in any parasite stage. This indicates that PfNapL is exclusively
located in the cytoplasm of the parasite and that its putative NES is not
necessary for its localization, and the predicted NES is not
functional/relevant in PfNapL.</p><p><fig position="float" id="fig3"><label>FIGURE 3.</label><caption><p><bold>NES and NLS motifs in yNAP-1 and PfNapL.</bold> <italic>a</italic>, surface
representation of yNAP-1. NES, accessory domain, and NLS are <italic>colored red,
yellow</italic>, and <italic>magenta</italic>, respectively. <italic>b</italic>, surface
representation of PfNapL. Predicted NES is colored <italic>red</italic>. The predicted
NLS is indicated. <italic>c</italic>, sequence alignment of NES and NLS in yNAP-1 and
PfNapL. The key residues are <italic>colored green</italic>. Superimposition of NES in
yNAP-1 and PfNapL is shown with key residues shown in <italic>green</italic> as
<italic>ball-and-stick representations</italic>.</p></caption><graphic xlink:href="zbc0170971220003"></graphic></fig></p><p><fig position="float" id="fig4"><label>FIGURE 4.</label><caption><p><bold>Stage specific expression of PfNapL-GFP chimeras.</bold> <italic>a</italic>,
Western blot of transgenic cell lines expressing GFP-tagged versions of NapL
with antibodies against GFP. Expression of PfHsp70 was detected to ensure
similar loading. The localization of PfNapL wild type-GFP (<italic>b</italic>), PfNapL
L46A-GFP (<italic>c</italic>), and PfNapL Q50A (<italic>d</italic>) in different stages of the
erythrocytic life cycle of <italic>P. falciparum</italic> is shown. The <italic>first
column</italic> of each <italic>panel</italic> shows bright field pictures, followed by a
nuclear DAPI stain, GFP fluorescence, an overlay of the nuclear and GFP
localization, and (in the <italic>last column</italic>) overlay of bright field with
DAPI and GFP.</p></caption><graphic xlink:href="zbc0170971220004"></graphic></fig></p><p><italic>The Electrostatic Potential of PfNapL Dimer</italic>—Analysis of the
electrostatic potential of PfNapL dimer indicates that the convex region of
the dimerization helix α2 has acidic residues scattered on the surface
in an alternate manner (Glu<sup>38</sup>, Glu<sup>41</sup>, Glu<sup>52</sup>,
Asp<sup>55</sup>, Glu<sup>63</sup>, Asp<sup>80</sup>, and Glu<sup>84</sup>)
(<xref rid="fig5" ref-type="fig">Fig. 5</xref>). There are also three
basic residues toward the end of this helix (Lys<sup>70</sup>,
Lys<sup>81</sup>, and Arg<sup>82</sup>) contributing to a slightly basic
nature and hence an uneven charge distribution
(<xref rid="fig5" ref-type="fig">Fig. 5</xref>). The domain II of
PfNapL dimer has a hydrophobic nature on one side and is highly negatively
charged on the opposite face with several residues (Glu<sup>119</sup>,
Glu<sup>122</sup>, Glu<sup>123</sup>, Glu<sup>252</sup>, Glu<sup>256</sup>,
Asp<sup>258</sup>, Glu<sup>260</sup>, and Glu<sup>267</sup>), forming a cavity
that is also a characteristic feature of the yNAP-1 and hSET
(<xref rid="fig5" ref-type="fig">Fig. 5</xref>). The bottom of this
cavity has an uneven charge character, and a single acidic residue,
Glu<sup>61</sup>, is present at its base. It has been previously suggested for
yNAP-1 that this highly acidic cavity might be important for binding to basic
histones (<xref ref-type="bibr" rid="ref15">15</xref>). The residues
Asp<sup>119</sup>, Glu<sup>122</sup>, Glu<sup>123</sup>, Asp<sup>258</sup>,
Glu<sup>260</sup>, and Glu<sup>267</sup> that constitute this cavity in PfNapL
are structurally conserved with yNAP-1 and hSET, hence highlighting their
possible significance.</p><p><italic>Prediction of Essential Residues Involved in Histone Binding by
PfNapL</italic>—Most histone chaperones contain long acidic stretches in
their C-terminal region, which were thought to be involved in histone binding,
considering the highly basic character of histones. However, recent studies
have shown that these acidic stretches are not essential for histone binding
activities for chaperones yNAP-1, Asf1, nucleoplasmin, etc.
(<xref ref-type="bibr" rid="ref5">5</xref>,
<xref ref-type="bibr" rid="ref15">15</xref>,
<xref ref-type="bibr" rid="ref37">37</xref>). It is noted that
full-length PfNapL also has an acidic stretch of residues in its C-terminal
region similar to yNAP-1 and hSET, with an average pI of ∼3.0 calculated
using amino acid sequence (<xref rid="fig1" ref-type="fig">Fig.
1<italic>a</italic></xref>). Mutagenesis studies on hSET revealed important
residues of domain II that affect the binding of hSET to both core histones
and double-stranded DNA (<xref ref-type="bibr" rid="ref16">16</xref>).
We have mapped these residues on yNAP-1 and PfNapL
(<xref rid="fig6" ref-type="fig">Fig. 6<italic>a</italic></xref>). Based upon
these hSET mutagenesis data, we identified several corresponding residues in
PfNapL and mutated them to test their relevance in histone recognition. We
constructed six single-site mutants of the corresponding residues from PfNapL
(D192A, H227A, T230A, Y259A, E260A, and K266A). In addition, we made one other
mutant (D223A) that is conserved, exposed, and proximal to the previously
implicated histone recognition residues from hSET
(<xref ref-type="bibr" rid="ref16">16</xref>). Our PfNapL-histone
binding data, using purified proteins of high quality, show that none of these
seven mutations altered histone binding of PfNapL to histones H3, H4, H2A, or
H2B, to histone tetramer, or to histone octamer significantly
(<xref rid="fig6" ref-type="fig">Fig. 6<italic>b</italic></xref>). We
therefore propose that it is likely for histones to have different a binding
site(s) on PfNapL (and possibly on other NAPs) in comparison with hSET-histone
interactions (<xref ref-type="bibr" rid="ref16">16</xref>). These
residues are not identical among PfNapL, hSET, yNAP-1, and Vps75 proteins, and
in general, residues proposed by hSET-histone studies show weak conservation
(Table S1). An overall structure-based residue comparison between yNAP-1,
Vps75, hSET, and PfNapL reveals very few regions of sequence conservation
(Fig. S1). In order to further probe the potential sites for histone
recognition by PfNapL, we inspected the surface residues of PfNapL (keeping in
view structure and sequence information from NAPs). Here, we have highlighted
the identical/conserved surface residues in the NAP family (Figs.
<xref rid="fig1" ref-type="fig">1<italic>d</italic></xref> and
<xref rid="fig7" ref-type="fig">7</xref>) based on the described
criterion.</p><p><fig position="float" id="fig5"><label>FIGURE 5.</label><caption><p><bold>Electrostatic potential distribution of the PfNapL dimer.</bold> The convex
region of the dimerization helix α2 contains acidic residues scattered
on the surface in an alternate manner. Domain II of PfNapL forms a cavity
consisting of mostly acidic residues that are conserved with yNAP-1 and
hSET.</p></caption><graphic xlink:href="zbc0170971220005"></graphic></fig></p><p><italic>Comparisons with Asf1-Histone Complex</italic>—The β subdomain
of PfNapL is a highly conserved feature of NAPs and is also found in other
histone chaperones like Asf1
(<xref ref-type="bibr" rid="ref20">20</xref>). The crystal structure of
the human Asf1 has been recently determined in complex with H3-H4 dimer,
highlighting the crucial residues involved in histone binding
(<xref ref-type="bibr" rid="ref20">20</xref>,
<xref ref-type="bibr" rid="ref21">21</xref>). A structural comparison
of the four-stranded β subdomain of PfNapL with Asf1 shows a
superimposition with an r.m.s. deviation of 0.97 Å
(<xref rid="fig8" ref-type="fig">Fig. 8<italic>a</italic></xref>). Further,
there are no steric hindrances between the overall PfNapL dimer and H3-H4
dimer (Fig. S6). Mapping of the interacting residues as highlighted for human
Asf1 revealed three corresponding, structurally conserved surface residues in
PfNapL (Ile<sup>136</sup>, Ile<sup>147</sup>, and Ala<sup>185</sup>)
(<xref rid="fig8" ref-type="fig">Fig. 8<italic>b</italic></xref>). Further,
three residues of PfNapL are conserved among the NAP family
(<xref rid="fig8" ref-type="fig">Fig. 8<italic>c</italic></xref>) and may
represent common sites for histone recognition. Based on structural
congruence, we propose that these residues in PfNapL may interact with
Leu<sup>126</sup> and Ile<sup>130</sup> of H3 and Thr<sup>96</sup> and
Leu<sup>97</sup> of histone H4. To validate the above modeling, we mutated the
putative interacting PfNapL residues, Ile<sup>136</sup> and Ile<sup>147</sup>,
to alanines and tested the mutant proteins for H3-H4 tetramer binding ability.
Our protein-protein-based ELISA data indicate a significant (up to two-thirds)
reduction in binding between PfNapL and H3-H4 tetramer
(<xref rid="fig8" ref-type="fig">Fig. 8<italic>d</italic></xref>).</p></sec><sec><title>DISCUSSION</title><p>The structure of PfNapL was determined using the iodide-SAD technique and
refined to 2.3 Å resolution. Structural analysis of PfNapL has revealed
that PfNapL conforms to the domain architecture of nucleosome assembly
proteins, exhibiting a high complexity ordered central region and largely
disordered N- and C-terminal regions. PfNapL forms a homodimer that acquires a
fold similar to the yNAP-1, hSET, and Vps75 dimers
(<xref ref-type="bibr" rid="ref15">15</xref>-<xref ref-type="bibr" rid="ref17">17</xref>).
These investigations demonstrate high structural homology within these
proteins despite a low level of sequence identity (only 14-25%)
(<xref rid="fig1" ref-type="fig">Fig. 1<italic>c</italic></xref>). The major
difference between PfNapL and yNAP-1 lies in the absence of the accessory
domain in PfNapL that is inserted between the dimerization helix α2 and
domain II in yNAP-1 (<xref ref-type="bibr" rid="ref15">15</xref>)
(<xref rid="fig1" ref-type="fig">Fig. 1<italic>d</italic></xref>).
Phylogenetic analysis of NAPs suggests greater evolutionary distance of PfNapL
from homologs in higher eukaryotes (<xref rid="fig1" ref-type="fig">Fig.
1<italic>e</italic></xref>), although malaria parasite NAPs are unique in their
lack of the accessory domain. We have earlier shown that PfNapL is a unique,
nonredundant protein in <italic>P. falciparum</italic>, and it performs an exclusive
role in the nucleosome assembly activity in the parasite
(<xref ref-type="bibr" rid="ref13">13</xref>,
<xref ref-type="bibr" rid="ref14">14</xref>). Previous studies on gene
knock-out phenotypes in several organisms like mouse and <italic>Drosophila</italic>have indicated the essentiality of the NAP family
(<xref ref-type="bibr" rid="ref9">9</xref>,
<xref ref-type="bibr" rid="ref10">10</xref>). Furthermore, our
inability to disrupt the gene for PfNapL corroborates the essentiality of this
protein in <italic>P. falciparum</italic> (Fig. S2).</p><p><fig position="float" id="fig6"><label>FIGURE 6.</label><caption><p><italic>a</italic>, orthogonal views of the surface representation of hSET, yNAP-1,
and PfNapL dimers with important residues from the mutagenesis data of hSET
<italic>colored green</italic> and <italic>red. b</italic>, histone-binding activities of
PfNapL wild type and mutants (D192A, H227A, T230A, E260A, K266A, D233A, and
Y259A). Shown is binding with histone tetramer (<italic>i</italic>), histone octamer
(<italic>ii</italic>), H3 (<italic>iii</italic>), H4 (<italic>iv</italic>), H2A (<italic>v</italic>), and H2B
(<italic>vi</italic>).</p></caption><graphic xlink:href="zbc0170971220006"></graphic></fig></p><p>Corresponding to yNAP-1, PfNapL also contains NES and NLS motifs (Figs.
<xref rid="fig1" ref-type="fig">1<italic>c</italic></xref> and
<xref rid="fig3" ref-type="fig">3<italic>c</italic></xref>). We have
previously shown that PfNapL is predominantly localized to the cytoplasm and
is centrally responsible for shuttling and transfer of histones to PfNapS
(which is localized to both cytoplasm and the nucleus and which eventually
deposits histones onto DNA). PfNapL and PfNapS do not interact with each other
(<xref ref-type="bibr" rid="ref14">14</xref>). Therefore, PfNapL is
principally localized to the cytoplasm and does not possess the ability to
deposit the histones in the nucleus
(<xref ref-type="bibr" rid="ref14">14</xref>). In this context, it is
interesting to note that hSET also lacks the accessory domain of yNAP-1/other
NAPs, but unlike PfNapL, hSET is localized both in cytoplasm and the nucleus
(<xref ref-type="bibr" rid="ref22">22</xref>). Since this accessory
domain is absent in PfNapL (<xref rid="fig2" ref-type="fig">Fig.
2<italic>b</italic></xref>), the predicted NES on the PfNapL structure is thus
exposed and not be masked by the accessory domain, as suggested and observed
for yNAP-1 (<xref ref-type="bibr" rid="ref15">15</xref>)
(<xref rid="fig3" ref-type="fig">Fig. 3, <italic>a</italic> and
<italic>b</italic></xref>). From a structural perspective, this further validates
the localization of PfNapL to the cytoplasm. The histone binding
characteristics of PfNapL have been detailed previously, including its ability
to transfer cytoplasmic histones on to PfNapS. A model for relay of histones
from parasite cytoplasm to the nucleus has also been proposed by us previously
(see Ref. <xref ref-type="bibr" rid="ref14">14</xref> and specifically
<xref rid="fig1" ref-type="fig">Fig. 1<italic>f</italic></xref> reproduced
here from Ref. <xref ref-type="bibr" rid="ref14">14</xref>). Our
published reports on the two <italic>Plasmodium</italic> parasite nucleosome assembly
proteins have clearly suggested different roles for PfNapL (in histone
shuttling in the cytoplasm) and for PfNapS (which is localized both in the
cytoplasm and nucleus and which can perform histone deposition onto DNA
(<xref ref-type="bibr" rid="ref13">13</xref>,
<xref ref-type="bibr" rid="ref14">14</xref>)
(<xref rid="fig1" ref-type="fig">Fig. 1<italic>f</italic></xref>).</p><p><fig position="float" id="fig7"><label>FIGURE 7.</label><caption><p><bold>Two views of the surface representation of PfNapL and yNAP-1 dimers.</bold>Corresponding residues from the mutagenesis data of hSET are <italic>colored
green</italic> and <italic>red</italic>. The identical/conserved surface residues within
the NAP family are <italic>colored yellow</italic>.</p></caption><graphic xlink:href="zbc0170971220007"></graphic></fig></p><p>Electrostatic potential distribution of PfNapL dimer reveals a stretch of
alternate acidic residues on the convex surface of the dimerization helix
α2 and a highly acidic side to domain II. If this convex surface of the
PfNapL dimer is implicated in histone recognition, then it can be suggested
that the highlighted acidic residues on this surface might be making alternate
contacts with an α-helix of a core histone
(<xref rid="fig5" ref-type="fig">Fig. 5</xref>). In yNAP-1, the acidic
stretch forming a cavity in domain II has been shown to be involved in
neutralizing and binding the basic N-terminal histone tails
(<xref ref-type="bibr" rid="ref15">15</xref>). Also, most of the acidic
residues constituting this cavity are structurally conserved among PfNapL and
yNAP-1. Thus, based upon the earlier suggestions for yNAP-1, this acidic
region in PfNapL may be involved in histone recognition
(<xref rid="fig5" ref-type="fig">Fig. 5</xref>).</p><p>Mapping of the corresponding residues from hSET mutagenesis data onto
PfNapL and yNAP-1 demonstrates the conserved nature of 10 of these residues
among PfNapL, hSET, and yNAP-1 (<xref rid="fig6" ref-type="fig">Fig.
6<italic>a</italic></xref>). However, mutation of some of these residues on
PfNapL did not significantly alter PfNapL-histone binding
(<xref rid="fig6" ref-type="fig">Fig. 6<italic>b</italic></xref>).
Consequently, based upon our mutagenesis data analysis, we suggest that the
binding site(s) and/or essential residues for the recognition of core histones
may be different for PfNapL from those proposed by hSET-histone interactions
(<xref ref-type="bibr" rid="ref16">16</xref>). A structure-based
comparison of the residues that were mutated in PfNapL reveals that none of
these are identical among hSET, yNAP-1, Vps75, and PfNapL. Indeed, only a few
show weak conservation (Table S1). Also, an overall structure-based comparison
between yNAP-1, Vps75, hSET, and PfNapL reveals very few regions of sequence
conservation (Fig. S1). These observations therefore suggest that histone
recognition site(s) for hSET may not be applicable for nucleosome assembly
proteins like PfNapL and yNAP-1. Our mapping of highly conserved and
surface-exposed residues in the NAP family provides some clues for the
possible set of essential residues that might underpin NAP-histone binding
(Figs. <xref rid="fig1" ref-type="fig">1<italic>d</italic></xref> and
<xref rid="fig7" ref-type="fig">7</xref>). Several of these conserved
residues lie in and around the characteristic cavity formed by the PfNapL
dimer, which has been previously implicated in histone recognition in both
yNAP-1 and hSET (<xref ref-type="bibr" rid="ref15">15</xref>,
<xref ref-type="bibr" rid="ref16">16</xref>). Distribution of these
conserved surface residues on the convex region of PfNapL dimer illustrates
their proximity to and overlap with the alternate stretch of acidic residues
highlighted by our electrostatic potential analysis.</p><p>It is known that the β subdomain present in yNAP-1, hSET, and PfNapL
is a highly conserved feature of histone chaperones
(<xref rid="fig2" ref-type="fig">Fig. 2<italic>a</italic></xref>). The
superimposition of the β subdomain of PfNapL onto histone chaperone Asf1
revealed three conserved surface residues on PfNapL (conserved in all NAPs)
that are suggested to be important for interaction with H3-H4 dimer based upon
the Asf1 and H3-H4 dimer interactions
(<xref ref-type="bibr" rid="ref21">21</xref>)
(<xref rid="fig8" ref-type="fig">Fig. 8, <italic>a-d</italic></xref>, and Fig.
S6). Interestingly, the three residues identified in Asf-histone complex map
to a region of PfNapL that is not implicated by either hSET mutagenesis or by
our mapping of conserved, exposed residues in NAPs. This suggests that Asf1
and NAPs may recognize histones differently. Further, modeling of PfNapL based
on the Asf1-histone complex crystal structure clearly suggests three target
residues that are likely to participate in H3-H4 recognition. Based on these
structural insights, we mutated two of the three residues (the third one is an
alanine in any case) and confirmed the idea that their alteration to alanine
reduces binding of PfNapL to H3-H4 tetramer significantly
(<xref rid="fig8" ref-type="fig">Fig. 8<italic>d</italic></xref>).</p><p><fig position="float" id="fig8"><label>FIGURE 8.</label><caption><p><bold>Comparison of PfNapL dimer with the Asf1-histone complex (Protein Data
Bank code 2HUE).</bold> <italic>a</italic>, superimposition of the β subdomain of
PfNapL (<italic>purple ribbon</italic>) and Asf1-histone complex wherein Asf1, H3, and
H4 are <italic>colored light gray, pink</italic>, and <italic>brown</italic>, respectively.
The conserved residues of PfNapL are shown in a <italic>red ball-and-stick
representation</italic>. The interacting residues of H3 and H4 are shown in a
<italic>yellow</italic> and <italic>green ball-and-stick representation. b</italic>, close
view of the proposed interaction. <italic>c</italic>, surface representation of PfNapL
with predicted histone-binding residues <italic>colored red. d</italic>,
histone-binding activity of PfNapL wild type and mutants (I136A and I147A)
with H3-H4 tetramer.</p></caption><graphic xlink:href="zbc0170971220008"></graphic></fig></p><p>In summary, our studies provide further structural insights into histone
chaperones from <italic>P. falciparum</italic> and highlight key regions of
differences between plasmodial NAPs and their counterparts. Although further
dissection of the modes of histone recognition by NAPs will be required to
gain a complete understanding of the mechanism of histone binding and
transport by nucleosome assembly proteins, it is already clear that the
parasite NAPs are sufficiently different from their counterparts in other
species. Our genetic-structural-functional data on PfNapL suggests PfNapL as a
possible new focus for development of antimalarials.</p></sec><sec sec-type="supplementary-material"><title>Supplementary Material</title><supplementary-material id="PMC_1" content-type="local-data"><caption><title>[Supplemental Data]</title></caption><media mimetype="text" mime-subtype="html" xlink:href="M808633200_index.html"></media><media xlink:role="associated-file" mimetype="application" mime-subtype="pdf" xlink:href="M808633200_1.pdf"></media></supplementary-material></sec></body><back><ack><p>We thank Prof. Alan Cowman for support and for the generous gift of
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NAP-1; hSET, human SET; SAD, single anomalous dispersion; NES, nuclear export
signal; DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent
protein; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered
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