The CaMKII holoenzyme structure in activation-competent conformations
Identifieur interne : 000704 ( Pmc/Checkpoint ); précédent : 000703; suivant : 000705The CaMKII holoenzyme structure in activation-competent conformations
Auteurs : Janette B. Myers [États-Unis] ; Vincent Zaegel [États-Unis] ; Steven J. Coultrap [États-Unis] ; Adam P. Miller [États-Unis] ; K. Ulrich Bayer [États-Unis] ; Steve L. Reichow [États-Unis]Source :
- Nature Communications [ 2041-1723 ] ; 2017.
Abstract
The Ca2+/calmodulin-dependent protein kinase II (CaMKII) assembles into large 12-meric holoenzymes, which is thought to enable regulatory processes required for synaptic plasticity underlying learning, memory and cognition. Here we used single particle electron microscopy (EM) to determine a pseudoatomic model of the CaMKIIα holoenzyme in an extended and activation-competent conformation. The holoenzyme is organized by a rigid central hub complex, while positioning of the kinase domains is highly flexible, revealing dynamic holoenzymes ranging from 15–35 nm in diameter. While most kinase domains are ordered independently, ∼20% appear to form dimers and <3% are consistent with a compact conformation. An additional level of plasticity is revealed by a small fraction of bona-fide 14-mers (<4%) that may enable subunit exchange. Biochemical and cellular FRET studies confirm that the extended state of CaMKIIα resolved by EM is the predominant form of the holoenzyme, even under molecular crowding conditions.
Url:
DOI: 10.1038/ncomms15742
PubMed: 28589927
PubMed Central: 5467236
Affiliations:
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Myers</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Department of Chemistry, Portland State University</institution>, Portland, Oregon 97021,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Zaegel, Vincent" sort="Zaegel, Vincent" uniqKey="Zaegel V" first="Vincent" last="Zaegel">Vincent Zaegel</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Department of Pharmacology, University of Colorado</institution>, Aurora, Colorado 80045,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Coultrap, Steven J" sort="Coultrap, Steven J" uniqKey="Coultrap S" first="Steven J." last="Coultrap">Steven J. Coultrap</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Department of Pharmacology, University of Colorado</institution>, Aurora, Colorado 80045,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Miller, Adam P" sort="Miller, Adam P" uniqKey="Miller A" first="Adam P." last="Miller">Adam P. Miller</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Department of Chemistry, Portland State University</institution>, Portland, Oregon 97021,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Bayer, K Ulrich" sort="Bayer, K Ulrich" uniqKey="Bayer K" first="K. Ulrich" last="Bayer">K. Ulrich Bayer</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Department of Pharmacology, University of Colorado</institution>, Aurora, Colorado 80045,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Reichow, Steve L" sort="Reichow, Steve L" uniqKey="Reichow S" first="Steve L." last="Reichow">Steve L. Reichow</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>Department of Chemistry, Portland State University</institution>, Portland, Oregon 97021,<country>USA</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></analytic><series><title level="j">Nature Communications</title><idno type="eISSN">2041-1723</idno><imprint><date when="2017">2017</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The Ca<sup>2+</sup>/calmodulin-dependent protein kinase II (CaMKII) assembles into large 12-meric holoenzymes, which is thought to enable regulatory processes required for synaptic plasticity underlying learning, memory and cognition. Here we used single particle electron microscopy (EM) to determine a pseudoatomic model of the CaMKIIα holoenzyme in an extended and activation-competent conformation. The holoenzyme is organized by a rigid central hub complex, while positioning of the kinase domains is highly flexible, revealing dynamic holoenzymes ranging from 15–35 nm in diameter. While most kinase domains are ordered independently, ∼20% appear to form dimers and <3% are consistent with a compact conformation. An additional level of plasticity is revealed by a small fraction of bona-fide 14-mers (<4%) that may enable subunit exchange. Biochemical and cellular FRET studies confirm that the extended state of CaMKIIα resolved by EM is the predominant form of the holoenzyme, even under molecular crowding conditions.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Coultrap, S J" uniqKey="Coultrap S">S. J. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Nat Commun</journal-id><journal-id journal-id-type="iso-abbrev">Nat Commun</journal-id><journal-title-group><journal-title>Nature Communications</journal-title></journal-title-group><issn pub-type="epub">2041-1723</issn><publisher><publisher-name>Nature Publishing Group</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">28589927</article-id><article-id pub-id-type="pmc">5467236</article-id><article-id pub-id-type="pii">ncomms15742</article-id><article-id pub-id-type="doi">10.1038/ncomms15742</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>The CaMKII holoenzyme structure in activation-competent conformations</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Myers</surname><given-names>Janette B.</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="author-notes" rid="n1">*</xref></contrib><contrib contrib-type="author"><name><surname>Zaegel</surname><given-names>Vincent</given-names></name><xref ref-type="aff" rid="a2">2</xref><xref ref-type="author-notes" rid="n1">*</xref></contrib><contrib contrib-type="author"><name><surname>Coultrap</surname><given-names>Steven J.</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Miller</surname><given-names>Adam P.</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Bayer</surname><given-names>K. Ulrich</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Reichow</surname><given-names>Steve L.</given-names></name><xref ref-type="corresp" rid="c2">b</xref><xref ref-type="aff" rid="a1">1</xref></contrib><aff id="a1"><label>1</label><institution>Department of Chemistry, Portland State University</institution>, Portland, Oregon 97021,<country>USA</country></aff><aff id="a2"><label>2</label><institution>Department of Pharmacology, University of Colorado</institution>, Aurora, Colorado 80045,<country>USA</country></aff></contrib-group><author-notes><corresp id="c1"><label>a</label><email>ulli.bayer@ucdenver.edu</email></corresp><corresp id="c2"><label>b</label><email>reichow@pdx.edu</email></corresp><fn id="n1"><label>*</label><p>These authors contributed equally to this work.</p></fn></author-notes><pub-date pub-type="epub"><day>07</day><month>06</month><year>2017</year></pub-date><pub-date pub-type="collection"><year>2017</year></pub-date><volume>8</volume><elocation-id>15742</elocation-id><history><date date-type="received"><day>29</day><month>11</month><year>2016</year></date><date date-type="accepted"><day>25</day><month>04</month><year>2017</year></date></history><permissions><copyright-statement>Copyright © 2017, The Author(s)</copyright-statement><copyright-year>2017</copyright-year><copyright-holder>The Author(s)</copyright-holder><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/4.0/"><pmc-comment>author-paid</pmc-comment>
<license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link></license-p></license></permissions><abstract><p>The Ca<sup>2+</sup>/calmodulin-dependent protein kinase II (CaMKII) assembles into large 12-meric holoenzymes, which is thought to enable regulatory processes required for synaptic plasticity underlying learning, memory and cognition. Here we used single particle electron microscopy (EM) to determine a pseudoatomic model of the CaMKIIα holoenzyme in an extended and activation-competent conformation. The holoenzyme is organized by a rigid central hub complex, while positioning of the kinase domains is highly flexible, revealing dynamic holoenzymes ranging from 15–35 nm in diameter. While most kinase domains are ordered independently, ∼20% appear to form dimers and <3% are consistent with a compact conformation. An additional level of plasticity is revealed by a small fraction of bona-fide 14-mers (<4%) that may enable subunit exchange. Biochemical and cellular FRET studies confirm that the extended state of CaMKIIα resolved by EM is the predominant form of the holoenzyme, even under molecular crowding conditions.</p></abstract><abstract abstract-type="web-summary"><p>Ca<sup>2+</sup>/calmodulin-dependent protein kinase II (CaMKII) forms a 12 subunit holoenzyme central to synaptic plasticity. Here the authors report a 3D structure of the CaMKII holoenzyme in an activation-competent state obtained by single particle EM, and suggest a role for the intrinsically disordered linker domain in facilitating cooperative activation.</p></abstract></article-meta></front><floats-group><fig id="f1"><label>Figure 1</label><caption><title>An extended form of the CaMKIIα holoenzyme resolved by single particle EM.</title><p>(<bold>a</bold>) Diagram of the CaMKIIα domain architecture (numbering of human isoform). Inset, shows a secondary structure diagram of the CaMKIIα regulatory domain. The coil region (faded red) represents a region that is disordered in the absence of calmodulin<xref ref-type="bibr" rid="b21">21</xref>. The site of auto-phosphorylation (T286) involved in regulating autonomous activity is indicated. (<bold>b</bold>) SDS–polyacrylamide gel electrophoresis of purified human CaMKIIα stained with Coomassie blue migrating at the expected molecular weight (MW ∼50 kDa). (<bold>c</bold>) Electron micrograph of negatively stained CaMKIIα particles. Contrast of protein is white on a dark background. Individual particles are indicated by white circles. Scale bar=100 nm. (<bold>d</bold>) Enlarged view of individual particles with the hub complex (blue outline) and twelve kinase domains (yellow circle) indicated. Scale bar=25 nm. (<bold>e</bold>,<bold>f</bold>) Single particle averaging and image analysis. Scale bar=25 nm. (<bold>e</bold>) Representative 2D projection average of unmasked particles. The central hub complex is clearly defined (blue outline), while the radial kinase domains appear as a diffuse halo (yellow circle). (<bold>f</bold>) 2D variance map of data in <bold>e</bold>. A region of high variance (<italic>white pixels</italic>) corresponds with the blurred region observed in 2D class averages (yellow circle). (<bold>g</bold>) Representative 2D projection average using an applied image mask (75 Å outer radius). The sixfold symmetric hub complex is clearly defined (blue outline). (<bold>h</bold>) Representative 2D projection average with an applied mask to remove contribution of the hub domain during the alignment procedure (50 Å inner radius mask). Twelve kinase domains are visualized at an approximate radius of 24–28 nm (yellow circles). (<bold>i</bold>) Class average in <bold>h</bold> with applied sixfold rotational averaging indicates a pseudo-symmetric kinase domain organization. (<bold>j</bold>) Composite image of <bold>g</bold> and <bold>i</bold> with the identified hub complex (blue outline) and kinase domains (yellow outline) indicated.</p></caption><graphic xlink:href="ncomms15742-f1"></graphic></fig><fig id="f2"><label>Figure 2</label><caption><title>3D-reconstruction and pseudo-atomic model of the CaMKIIα holoenzyme.</title><p>(<bold>a</bold>) 3D-reconstruction of the CaMKIIα holoenzyme (grey transparent) refined to ∼20 Å resolution. A pseudo-atomic model of the dodecameric holoenzyme (coloured as in <xref ref-type="fig" rid="f1">Fig. 1a</xref>) was constructed using previously determined crystal structures corresponding to the dodecameric CaMKIIα hub complex (blue ribbon, PDBID 5IG3 (ref. <xref ref-type="bibr" rid="b22">22</xref>); residues 345–472) and isolated kinase/regulatory domain (yellow/red ribbon, PDBID 2VZ6 (ref. <xref ref-type="bibr" rid="b21">21</xref>); residues 13–300). Residues 301–344 were modelled as disordered linkers connecting each kinase domain to the nearest hub domain. Residues 274–314 correspond to the regulatory domain (red), containing a proximal auto-inhibitory segment and distal calmodulin-binding site. (<bold>b</bold>,<bold>c</bold>) Unmasked 2D class average and single particle images (as shown in <xref ref-type="fig" rid="f1">Fig. 1</xref>) with crystal structures of the hub complex (blue surface) and kinase/regulatory domain (yellow surface) fit into the EM densities. A yellow halo in <bold>b</bold> represents the diffuse positioning of kinase domains in the 2D class average. Scale bars=25 nm. (<bold>d</bold>,<bold>e</bold>) Enlarged view of the fit domains in <bold>b</bold>,<bold>c</bold> illustrating the proposed structural equilibrium and variable kinase domain arrangements observed in single particle images. Flexible linkers connecting individual kinase domains to the central hub complex are represented as grey dotted lines.</p></caption><graphic xlink:href="ncomms15742-f2"></graphic></fig><fig id="f3"><label>Figure 3</label><caption><title>Structural diversity of CaMKIIα kinase domain arrangements measured from single particle images.</title><p>(<bold>a</bold>) Histogram of measured radius for individual kinase domains. A Gaussian curve fit to the histogram distribution is shown (grey line). (<bold>b</bold>) Illustration and whisker plot representation of the distribution of kinase radius measurements in <bold>a</bold>. Kinase domains (yellow) are illustrated to represent the minimum, maximum and average distance from the hub domain complex (blue). (<bold>c</bold>) Histogram of measured distance separating neighbouring kinase domains (center-to-center). A Gaussian curve fit to the histogram distribution is shown (grey line). (<bold>d</bold>) Illustration and whisker plot representation of the distribution of kinase separation measurements in <bold>c</bold>. Kinase domains are illustrated to represent the minimum, maximum and average kinase separation distances. Inset, in <bold>a</bold>,<bold>c</bold> illustrate the distance measurement made using raw particle images. Whiskers in <bold>b</bold>,<bold>d</bold> indicate the minimum and maximum values, and boxes indicate the 25%, 50% and 75% values of the distribution. (<bold>e</bold>) Correlation map of kinase radius versus kinase separation distance. This analysis revealed no statistical correlation between these values (correlation coefficient=0.2). The average of the two parameters is shown in red. Grey and yellow shading indicates regions of the correlation map where kinase domain positioning is consistent with steric contact with the hub domain and/or kinase-kinase contact, respectively.</p></caption><graphic xlink:href="ncomms15742-f3"></graphic></fig><fig id="f4"><label>Figure 4</label><caption><title>Putative kinase domain pairing in the CaMKIIα holoenzyme observed by single particle EM.</title><p>(<bold>a</bold>) Left, Distribution of kinase domain pairing arrangements, based on measured inter-domain distances (as described in main text). Holoenzyme structures were categorized as being arranged with 0–6 kinase domain pairs. Right, Illustration representing holoenzyme structures with various kinase domain pairing arrangements. These structures (and other various arrangements of kinase pairing) are suggested to be in equilibrium with various unpaired kinase arrangements. (<bold>b</bold>) Crystallographic structure of the <italic>C</italic>. <italic>elegans</italic> CaMKIIα kinase/regulatory domain (yellow/red ribbon; PDBID 2BDW (ref. <xref ref-type="bibr" rid="b19">19</xref>)) previously shown to form a dimeric interface involving the regulatory domain (<italic>red</italic>). (<bold>c</bold>) <italic>Left,</italic> Reference-free 2D class average observed for a small population of CaMKIIα particles apparently organized with all kinase domains forming paired interactions. <italic>Right,</italic> Crystal structures of the hub complex (blue surface) and dimeric kinase domains (yellow surface) are fit into the EM densities. Scale bar=25 nm.</p></caption><graphic xlink:href="ncomms15742-f4"></graphic></fig><fig id="f5"><label>Figure 5</label><caption><title>Full-length CaMKIIα wild type expressed in eukaryotic cells remains largely in an extended conformation even during molecular crowding.</title><p>(<bold>a</bold>) Models of the compact versus extended CaMKII holoenzyme conformations, which differ in their accessibility to Ca<sup>2+</sup>/CaM. The compact conformation has been described for a linker-less (LL) CaMKII and suggested during molecular crowding also for wild type (WT) based on increased Hill slope; the I321E mutation prevented the compact conformation even for the LL mutant<xref ref-type="bibr" rid="b23">23</xref>. (<bold>b</bold>) Ca<sup>2+</sup>/CaM dose/response curves for <italic>in vitro</italic> activation of rodent CaMKII without crowding (Contr) or with crowding by 150 mg ml<sup>−1</sup> BSA or lysozyme (lys). The curve fits shown are based on data from two independent experiments; the curve fits from the individual experiments are shown <xref ref-type="supplementary-material" rid="S1">Supplementary Fig. 6</xref>. (<bold>c</bold>) The Hill slope was not increased by molecular crowding and thus gave no indication for induction of a compact conformation. By contrast, the EC<sub>50</sub> was increased dramatically by lysozyme and to a lesser extent by BSA. (<bold>d</bold>) Human CaMKIIα and its I321E mutant that is incompetent for the compact conformation showed the same Ca<sup>2+</sup>/CaM responses during crowding with BSA as rodent CaMKIIα. (<bold>e</bold>,<bold>f</bold>) Linker-less (LL) CaMKII and its I321E mutant showed the same Ca<sup>2+</sup>/CaM response without crowding. However, crowding with BSA caused a lesser increase in E<sub>50</sub> for the I321E mutant that is incompetent for the compact conformation. The Hill slope was identical for both linker-less mutants and no longer showed significant cooperativity under crowding conditions. Error bars represent the s.e. calculated from the curve fits.</p></caption><graphic xlink:href="ncomms15742-f5"></graphic></fig><fig id="f6"><label>Figure 6</label><caption><title>A FRET-based assay indicated a compact conformation within cells only for linker-less but not for full-length CaMKIIα wild type.</title><p>(<bold>a</bold>) Co-expression of CaMKIIα labelled with mCherry or mGFP (as FRET donor or acceptor) N-terminal of the kinase domain resulted in a significant FRET signal in HEK cells (FRETc; corrected for fluorescence bleed-through). Deletion of the hub domain in the GFP-labelled CaMKII (1–316; <italic>n</italic>=12 cells) abolished the FRET signal almost completely. Scale bar=10 μm. (<bold>b</bold>) FRETc was normalized by expression levels of the FRET donor (FRETc/donor) and plotted as function of the acceptor/donor ratio (within a range of 4–11 fold acceptor access). The few cells that showed complete FRET failure (grey) remained included in the analysis. (<bold>c</bold>) The linker-less (LL) CaMKIIα mutant (<italic>n</italic>=17 cells) showed significantly higher FRET than all other constructs (***<italic>P</italic><0.001; ANOVA with Tukeys post-hoc analysis). FRET of full-length CaMKIIα wild type (WT; <italic>n</italic>=38), of its I321E mutant (full I321E; <italic>n</italic>=26) that is incompetent for the compact conformation, and of a linker-less I321E mutant (LL I321E; <italic>n</italic>=22) were indistinguishable (NS: <italic>P</italic>>0.05). Error bars represent the s.e.m.</p></caption><graphic xlink:href="ncomms15742-f6"></graphic></fig><fig id="f7"><label>Figure 7</label><caption><title>Overview of CaMKIIα kinase domain arrangements.</title><p>(<bold>a</bold>) Illustration of the CaMKII holoenzyme indicating an overall volume of kinase domain occupancy observed by single particle EM (yellow halo). Flexible linker regions support a continuum of kinase domain arrangements within this volume of occupancy (maximum diameter of ∼35 nm). (<bold>b</bold>) Three major conformational states appear to exist in equilibrium. A predominant extended and activatable state, as depicted in <bold>a</bold>, is distinguished by an extended conformation with non-interacting kinase domains. Additional non-activatable states are distinguished by the presence of dimeric pairing between neighbouring kinase domains (representing <20% of subunits), as well as a putative compact form distinguished by kinase-hub domain interactions (representing <3% of subunits). For clarity, fully paired and fully compact states are illustrated in <bold>b</bold>. However, conformational states where all twelve kinase domains are simultaneously paired appear to be rare (∼2.5% of structures) and the fully compact state with all kinase subunits of an individual holoenzyme in the compact conformation was not observed at all for full-length CaMKII holoenzymes by EM or by live cell FRET analysis.</p></caption><graphic xlink:href="ncomms15742-f7"></graphic></fig></floats-group></pmc><affiliations><list><country><li>États-Unis</li></country></list><tree><country name="États-Unis"><noRegion><name sortKey="Myers, Janette B" sort="Myers, Janette B" uniqKey="Myers J" first="Janette B." last="Myers">Janette B. Myers</name></noRegion><name sortKey="Bayer, K Ulrich" sort="Bayer, K Ulrich" uniqKey="Bayer K" first="K. Ulrich" last="Bayer">K. Ulrich Bayer</name><name sortKey="Coultrap, Steven J" sort="Coultrap, Steven J" uniqKey="Coultrap S" first="Steven J." last="Coultrap">Steven J. Coultrap</name><name sortKey="Miller, Adam P" sort="Miller, Adam P" uniqKey="Miller A" first="Adam P." last="Miller">Adam P. Miller</name><name sortKey="Reichow, Steve L" sort="Reichow, Steve L" uniqKey="Reichow S" first="Steve L." last="Reichow">Steve L. Reichow</name><name sortKey="Zaegel, Vincent" sort="Zaegel, Vincent" uniqKey="Zaegel V" first="Vincent" last="Zaegel">Vincent Zaegel</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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