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• W2010966202 abstract "•NMR, mutagenesis, and modeling identify substrate-binding sites in chaperonin TRiC•Subunit-specific polar/hydrophobic patterns define substrate-binding specificity•TRiC subunit diversification enables combinatorial substrate-recognition code•TRiC-bound substrate topology determined by subunit-specific motif recognition The eukaryotic chaperonin TRiC (also called CCT) is the obligate chaperone for many essential proteins. TRiC is hetero-oligomeric, comprising two stacked rings of eight different subunits each. Subunit diversification from simpler archaeal chaperonins appears linked to proteome expansion. Here, we integrate structural, biophysical, and modeling approaches to identify the hitherto unknown substrate-binding site in TRiC and uncover the basis of substrate recognition. NMR and modeling provided a structural model of a chaperonin-substrate complex. Mutagenesis and crosslinking-mass spectrometry validated the identified substrate-binding interface and demonstrate that TRiC contacts full-length substrates combinatorially in a subunit-specific manner. The binding site of each subunit has a distinct, evolutionarily conserved pattern of polar and hydrophobic residues specifying recognition of discrete substrate motifs. The combinatorial recognition of polypeptides broadens the specificity of TRiC and may direct the topology of bound polypeptides along a productive folding trajectory, contributing to TRiC’s unique ability to fold obligate substrates. The eukaryotic chaperonin TRiC (also called CCT) is the obligate chaperone for many essential proteins. TRiC is hetero-oligomeric, comprising two stacked rings of eight different subunits each. Subunit diversification from simpler archaeal chaperonins appears linked to proteome expansion. Here, we integrate structural, biophysical, and modeling approaches to identify the hitherto unknown substrate-binding site in TRiC and uncover the basis of substrate recognition. NMR and modeling provided a structural model of a chaperonin-substrate complex. Mutagenesis and crosslinking-mass spectrometry validated the identified substrate-binding interface and demonstrate that TRiC contacts full-length substrates combinatorially in a subunit-specific manner. The binding site of each subunit has a distinct, evolutionarily conserved pattern of polar and hydrophobic residues specifying recognition of discrete substrate motifs. The combinatorial recognition of polypeptides broadens the specificity of TRiC and may direct the topology of bound polypeptides along a productive folding trajectory, contributing to TRiC’s unique ability to fold obligate substrates. The health and integrity of the cellular proteome depend on molecular chaperones, which through their distinct substrate specificities and modes of action maintain protein homeostasis (Balch et al., 2008Balch W.E. Morimoto R.I. Dillin A. Kelly J.W. Adapting proteostasis for disease intervention.Science. 2008; 319: 916-919Crossref PubMed Scopus (1748) Google Scholar, Kim et al., 2013Kim Y.E. Hipp M.S. Bracher A. Hayer-Hartl M. Hartl F.U. Molecular chaperone functions in protein folding and proteostasis.Annu. Rev. Biochem. 2013; 82: 323-355Crossref PubMed Scopus (973) Google Scholar, Li and Buchner, 2013Li J. Buchner J. Structure, function and regulation of the hsp90 machinery.Biom. J. 2013; 36: 106-117Crossref Scopus (311) Google Scholar, Saibil, 2013Saibil H. Chaperone machines for protein folding, unfolding and disaggregation.Nat. Rev. Mol. Cell Biol. 2013; 14: 630-642Crossref PubMed Scopus (657) Google Scholar). Among these, the eukaryotic chaperonin TRiC (for TCP-1 ring complex, also called CCT for chaperonin containing TCP1) is distinguished by its complex architecture and mechanism, which allow it to fold a subset of essential and topologically complex proteins, including cell-cycle regulators, signaling proteins, and cytoskeletal components (Bigotti and Clarke, 2008Bigotti M.G. Clarke A.R. Chaperonins: The hunt for the Group II mechanism.Arch. Biochem. Biophys. 2008; 474: 331-339Crossref PubMed Scopus (50) Google Scholar, Kim et al., 2013Kim Y.E. Hipp M.S. Bracher A. Hayer-Hartl M. Hartl F.U. Molecular chaperone functions in protein folding and proteostasis.Annu. Rev. Biochem. 2013; 82: 323-355Crossref PubMed Scopus (973) Google Scholar). TRiC/CCT is a large hetero-oligomeric ATP-dependent complex consisting of two eight-membered rings stacked back to back (Bigotti and Clarke, 2008Bigotti M.G. Clarke A.R. Chaperonins: The hunt for the Group II mechanism.Arch. Biochem. Biophys. 2008; 474: 331-339Crossref PubMed Scopus (50) Google Scholar, Hartl et al., 2011Hartl F.U. Bracher A. Hayer-Hartl M. Molecular chaperones in protein folding and proteostasis.Nature. 2011; 475: 324-332Crossref PubMed Scopus (2190) Google Scholar, Spiess et al., 2004Spiess C. Meyer A.S. Reissmann S. Frydman J. Mechanism of the eukaryotic chaperonin: protein folding in the chamber of secrets.Trends Cell Biol. 2004; 14: 598-604Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Each ring creates a central chamber where substrate polypeptides bind and fold. Unlike simpler archaeal chaperonins, TRiC contains eight different paralogous subunits, named CCT1–CCT8, at fixed positions within each ring (Kalisman et al., 2012Kalisman N. Adams C.M. Levitt M. Subunit order of eukaryotic TRiC/CCT chaperonin by cross-linking, mass spectrometry, and combinatorial homology modeling.Proc. Natl. Acad. Sci. USA. 2012; 109: 2884-2889Crossref PubMed Scopus (127) Google Scholar, Leitner et al., 2012Leitner A. Joachimiak L.A. Bracher A. Mönkemeyer L. Walzthoeni T. Chen B. Pechmann S. Holmes S. Cong Y. Ma B. et al.The molecular architecture of the eukaryotic chaperonin TRiC/CCT.Structure. 2012; 20: 814-825Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). All subunits are structural homologs that consist of an ATP-binding equatorial domain and a substrate-binding apical domain linked by an intermediate domain (Bigotti and Clarke, 2008Bigotti M.G. Clarke A.R. Chaperonins: The hunt for the Group II mechanism.Arch. Biochem. Biophys. 2008; 474: 331-339Crossref PubMed Scopus (50) Google Scholar, Spiess et al., 2004Spiess C. Meyer A.S. Reissmann S. Frydman J. Mechanism of the eukaryotic chaperonin: protein folding in the chamber of secrets.Trends Cell Biol. 2004; 14: 598-604Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar) (Figure 1A). Each subunit also contains an apical segment that forms a lid over the cavity. An ATP-driven conformational cycle links TRiC-mediated folding to opening and closure of the lid, encapsulating the substrate in the cavity (Cong et al., 2012Cong Y. Schröder G.F. Meyer A.S. Jakana J. Ma B. Dougherty M.T. Schmid M.F. Reissmann S. Levitt M. Ludtke S.L. et al.Symmetry-free cryo-EM structures of the chaperonin TRiC along its ATPase-driven conformational cycle.EMBO J. 2012; 31: 720-730Crossref PubMed Scopus (61) Google Scholar, Meyer et al., 2003Meyer A.S. Gillespie J.R. Walther D. Millet I.S. Doniach S. Frydman J. Closing the folding chamber of the eukaryotic chaperonin requires the transition state of ATP hydrolysis.Cell. 2003; 113: 369-381Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, Reissmann et al., 2007Reissmann S. Parnot C. Booth C.R. Chiu W. Frydman J. Essential function of the built-in lid in the allosteric regulation of eukaryotic and archaeal chaperonins.Nat. Struct. Mol. Biol. 2007; 14: 432-440Crossref PubMed Scopus (86) Google Scholar, Reissmann et al., 2012Reissmann S. Joachimiak L.A. Chen B. Meyer A.S. Nguyen A. Frydman J. A gradient of ATP affinities generates an asymmetric power stroke driving the chaperonin TRIC/CCT folding cycle.Cell Rep. 2012; 2: 866-877Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Understanding how TRiC recognizes its substrates has important implications for human health (Balch et al., 2008Balch W.E. Morimoto R.I. Dillin A. Kelly J.W. Adapting proteostasis for disease intervention.Science. 2008; 319: 916-919Crossref PubMed Scopus (1748) Google Scholar). TRiC interacts with approximately 10% of the proteome and is essential for viability (Yam et al., 2008Yam A.Y. Xia Y. Lin H.T. Burlingame A. Gerstein M. Frydman J. Defining the TRiC/CCT interactome links chaperonin function to stabilization of newly made proteins with complex topologies.Nat. Struct. Mol. Biol. 2008; 15: 1255-1262Crossref PubMed Scopus (264) Google Scholar). Mutations in CCT5 and CCT4 are linked to sensory neuropathy (Bouhouche et al., 2006Bouhouche A. Benomar A. Bouslam N. Chkili T. Yahyaoui M. Mutation in the epsilon subunit of the cytosolic chaperonin-containing t-complex peptide-1 (Cct5) gene causes autosomal recessive mutilating sensory neuropathy with spastic paraplegia.J. Med. Genet. 2006; 43: 441-443Crossref PubMed Scopus (101) Google Scholar). Cancer-linked proteins p53, von Hippel Lindau tumor suppressor (VHL), and STAT3 are also TRiC substrates (Kasembeli et al., 2014Kasembeli M. Lau W.C. Roh S.H. Eckols T.K. Frydman J. Chiu W. Tweardy D.J. Modulation of STAT3 folding and function by TRiC/CCT chaperonin.PLoS Biol. 2014; 12: e1001844Crossref PubMed Google Scholar, Trinidad et al., 2013Trinidad A.G. Muller P.A. Cuellar J. Klejnot M. Nobis M. Valpuesta J.M. Vousden K.H. Interaction of p53 with the CCT complex promotes protein folding and wild-type p53 activity.Mol. Cell. 2013; 50: 805-817Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), and mutations in the TRiC-binding sites of VHL lead to misfolding tumorigenesis (Feldman et al., 1999Feldman D.E. Thulasiraman V. Ferreyra R.G. Frydman J. Formation of the VHL-elongin BC tumor suppressor complex is mediated by the chaperonin TRiC.Mol. Cell. 1999; 4: 1051-1061Abstract Full Text Full Text PDF PubMed Google Scholar, Feldman et al., 2003Feldman D.E. Spiess C. Howard D.E. Frydman J. Tumorigenic mutations in VHL disrupt folding in vivo by interfering with chaperonin binding.Mol. Cell. 2003; 12: 1213-1224Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). TRiC also suppresses aggregation and toxicity of Huntingtin in Huntington’s disease (Behrends et al., 2006Behrends C. Langer C.A. Boteva R. Böttcher U.M. Stemp M.J. Schaffar G. Rao B.V. Giese A. Kretzschmar H. Siegers K. Hartl F.U. Chaperonin TRiC promotes the assembly of polyQ expansion proteins into nontoxic oligomers.Mol. Cell. 2006; 23: 887-897Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, Kitamura et al., 2006Kitamura A. Kubota H. Pack C.G. Matsumoto G. Hirayama S. Takahashi Y. Kimura H. Kinjo M. Morimoto R.I. Nagata K. Cytosolic chaperonin prevents polyglutamine toxicity with altering the aggregation state.Nat. Cell Biol. 2006; 8: 1163-1170Crossref PubMed Scopus (219) Google Scholar, Tam et al., 2006Tam S. Geller R. Spiess C. Frydman J. The chaperonin TRiC controls polyglutamine aggregation and toxicity through subunit-specific interactions.Nat. Cell Biol. 2006; 8: 1155-1162Crossref PubMed Scopus (229) Google Scholar, Tam et al., 2009Tam S. Spiess C. Auyeung W. Joachimiak L. Chen B. Poirier M.A. Frydman J. The chaperonin TRiC blocks a huntingtin sequence element that promotes the conformational switch to aggregation.Nat. Struct. Mol. Biol. 2009; 16: 1279-1285Crossref PubMed Scopus (181) Google Scholar). TRiC is also important for folding viral proteins and required for replication of important human pathogens, including HCV and HIV (Inoue et al., 2011Inoue Y. Aizaki H. Hara H. Matsuda M. Ando T. Shimoji T. Murakami K. Masaki T. Shoji I. Homma S. et al.Chaperonin TRiC/CCT participates in replication of hepatitis C virus genome via interaction with the viral NS5B protein.Virology. 2011; 410: 38-47Crossref PubMed Scopus (50) Google Scholar, Zhou et al., 2008Zhou H. Xu M. Huang Q. Gates A.T. Zhang X.D. Castle J.C. Stec E. Ferrer M. Strulovici B. Hazuda D.J. Espeseth A.S. Genome-scale RNAi screen for host factors required for HIV replication.Cell Host Microbe. 2008; 4: 495-504Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar). In HIV, TRiC interacts with proteins Gag, Vif, and p6 (Hong et al., 2001Hong S. Choi G. Park S. Chung A.S. Hunter E. Rhee S.S. Type D retrovirus Gag polyprotein interacts with the cytosolic chaperonin TRiC.J. Virol. 2001; 75: 2526-2534Crossref PubMed Scopus (51) Google Scholar, Jäger et al., 2012Jäger S. Cimermancic P. Gulbahce N. Johnson J.R. McGovern K.E. Clarke S.C. Shales M. Mercenne G. Pache L. Li K. et al.Global landscape of HIV-human protein complexes.Nature. 2012; 481: 365-370Crossref Scopus (511) Google Scholar). The unique architecture and mechanistic features of TRiC set it apart from other chaperones. The diversification of subunits in TRiC is likely central to understand why many essential proteins, such as actin, Cdc20, and Cdh1, can only be folded with assistance from TRiC (Hartl et al., 2011Hartl F.U. Bracher A. Hayer-Hartl M. Molecular chaperones in protein folding and proteostasis.Nature. 2011; 475: 324-332Crossref PubMed Scopus (2190) Google Scholar, Spiess et al., 2004Spiess C. Meyer A.S. Reissmann S. Frydman J. Mechanism of the eukaryotic chaperonin: protein folding in the chamber of secrets.Trends Cell Biol. 2004; 14: 598-604Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Despite their extensive conservation in the ATP-binding domains, TRiC subunits have widely divergent functions within the ATP-driven cycle (Reissmann et al., 2012Reissmann S. Joachimiak L.A. Chen B. Meyer A.S. Nguyen A. Frydman J. A gradient of ATP affinities generates an asymmetric power stroke driving the chaperonin TRIC/CCT folding cycle.Cell Rep. 2012; 2: 866-877Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Additionally, the surface properties of the different subunits result in an asymmetric distribution of electrostatic charges within the folding chamber (Leitner et al., 2012Leitner A. Joachimiak L.A. Bracher A. Mönkemeyer L. Walzthoeni T. Chen B. Pechmann S. Holmes S. Cong Y. Ma B. et al.The molecular architecture of the eukaryotic chaperonin TRiC/CCT.Structure. 2012; 20: 814-825Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). The principles driving TRiC substrate recognition are poorly understood. In vivo, TRiC folds a subset of cellular proteins, suggesting a degree of specificity; however, its substrates are functionally and structurally diverse, indicating the potential to bind a broad array of proteins. The apical domains of each TRiC subunit are thought to recognize different motifs in substrates (Spiess et al., 2004Spiess C. Meyer A.S. Reissmann S. Frydman J. Mechanism of the eukaryotic chaperonin: protein folding in the chamber of secrets.Trends Cell Biol. 2004; 14: 598-604Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, Spiess et al., 2006Spiess C. Miller E.J. McClellan A.J. Frydman J. Identification of the TRiC/CCT substrate binding sites uncovers the function of subunit diversity in eukaryotic chaperonins.Mol. Cell. 2006; 24: 25-37Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) (Figures 1A and 1B). However, to date, no precise structural or sequence rules for TRiC-substrate binding have been identified. We here integrate biophysical and computational structural biology approaches with chemical crosslinking and mass spectrometry (XL-MS) to define the basis of TRiC-substrate recognition. We find that unique subunit-specific patterns of polar and hydrophobic residues underlie the distinct substrate binding properties of each subunit in the complex. The diversification of TRiC subunits thus provides a modular menu of binding specificities that allows for combinatorial recognition of substrate polypeptides. This likely contributes to TRiC’s unique ability to fold structurally diverse and topologically complex substrates. Evolutionary analyses further suggest that diversification of TRiC subunits from its simpler archaeal ancestors enabled the expansion of eukaryotic genomes to acquire proteins with novel folds and functions. To understand the molecular basis of this recognition specificity, we exploited substrates where the cognate CCT subunit and the relevant substrate motif have been identified (Figure 1C). The 54 amino acid-long HIV protein p6, and the related protein p4 from MPMV, associate directly with subunit CCT3 of TRiC (Hong et al., 2001Hong S. Choi G. Park S. Chung A.S. Hunter E. Rhee S.S. Type D retrovirus Gag polyprotein interacts with the cytosolic chaperonin TRiC.J. Virol. 2001; 75: 2526-2534Crossref PubMed Scopus (51) Google Scholar). A short 6–9 amino acid-long hydrophobic motif in VHL, called Box1, contacts subunit CCT1 (Spiess et al., 2006Spiess C. Miller E.J. McClellan A.J. Frydman J. Identification of the TRiC/CCT substrate binding sites uncovers the function of subunit diversity in eukaryotic chaperonins.Mol. Cell. 2006; 24: 25-37Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Importantly, the isolated recombinant apical domains of each TRiC subunit retain the ability to bind substrates and substrate-derived motifs with the specificity of the same subunits within the intact complex (Spiess et al., 2006Spiess C. Miller E.J. McClellan A.J. Frydman J. Identification of the TRiC/CCT substrate binding sites uncovers the function of subunit diversity in eukaryotic chaperonins.Mol. Cell. 2006; 24: 25-37Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, Tam et al., 2006Tam S. Geller R. Spiess C. Frydman J. The chaperonin TRiC controls polyglutamine aggregation and toxicity through subunit-specific interactions.Nat. Cell Biol. 2006; 8: 1155-1162Crossref PubMed Scopus (229) Google Scholar, Tam et al., 2009Tam S. Spiess C. Auyeung W. Joachimiak L. Chen B. Poirier M.A. Frydman J. The chaperonin TRiC blocks a huntingtin sequence element that promotes the conformational switch to aggregation.Nat. Struct. Mol. Biol. 2009; 16: 1279-1285Crossref PubMed Scopus (181) Google Scholar). We used purified HIV-p6 (herein p6) and VHL-Box1 (herein Box1) to examine the association of TRiC apical domains of CCT1 (herein ApiCCT1) and CCT3 (ApiCCT3) (Spiess et al., 2006Spiess C. Miller E.J. McClellan A.J. Frydman J. Identification of the TRiC/CCT substrate binding sites uncovers the function of subunit diversity in eukaryotic chaperonins.Mol. Cell. 2006; 24: 25-37Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) with cognate and noncognate substrate-recognition motifs (Figure 1D). A surface plasmon resonance (SPR)-based assay measured association and dissociation kinetics for ApiCCT-substrate pairs (Figures 1E and 1F and Figure S1 available online). Binding kinetics of immobilized VHL-Box1 and HIV-p6 to their cognate and noncognate ApiCCT binding partners were monitored by SPR over a range of concentrations (Figures 1D–1F and S1A–S1D). Apparent association and dissociation rates (Figure 1E) and binding constants (Figure 1F) were calculated from the sensograms (Figure S1). These indicated that the ratio of association over dissociation rates, i.e., the overall affinity, was higher for the cognate ApiCCT-substrate pairs (Figure 1E), consistent with the specificity of these motifs for these subunits within the TRiC complex. The measured on-rates, determined at approximately 103 M−1 s−1, were markedly slower than diffusion-controlled binding (Figure 1E, blue bars) but consistent with the relatively slow substrate-binding kinetics of TRiC (Melki et al., 1997Melki R. Batelier G. Soulié S. Williams Jr., R.C. Cytoplasmic chaperonin containing TCP-1: structural and functional characterization.Biochemistry. 1997; 36: 5817-5826Crossref PubMed Google Scholar). Cognate interactions exhibited slower dissociation kinetics than noncognate interactions (Figures 1E and S1A–S1D). Both association and dissociation rates contribute to substrate specificity for different subunits. For p6, the difference between cognate and noncognate interaction was largely driven by dissociation rates, whereas for Box1, cognate and noncognate discrimination was a result of differential on- and off-rates (Figure 1E). Of note, even the cognate interactions are relatively weak, with an overall affinity of approximately 0.25–0.5 μM (Figure 1F). Accordingly, stable TRiC binding to most substrates will depend on multivalent recognition of several elements in the polypeptide by several subunits in the chaperonin. We focused on the ApiCCT3 and p6 interaction pair to gain a deeper structural understanding of TRiC-substrate recognition. NMR-based chemical shift (CS) mapping was used to identify the substrate-recognition interface in ApiCCT3 (Figures 2 and S2). The 15N-1H Heteronuclear Single Quantum Coherence (HSQC) spectrum of ApiCCT3 yielded well-resolved and dispersed spectra, accounting for 142 of 167 peaks, covering 85% of the protein sequence (Figures 2A and S2A and not shown). Standard triple-resonance backbone experiments, guided by specific amino acid labeling to anchor the sequence connectivities allowed us to successfully assign >85% of the peaks in the 2D HSQC spectrum, including all the ApiCCT3 residues perturbed upon substrate addition (Figures S2A–S2D). Titration of increasing amounts of unlabeled p6 into 15N-labeled ApiCCT3 produced concentration-dependent shifts in a specific subset of peaks (Figures 2A and 2B); five peaks were strongly perturbed (>0.2 ppm), and another four peaks were perturbed weakly (>0.1 ppm; Figure 2B). Similar experiments were performed with p6-related protein p4 from M-PMV, which binds CCT3 with lower affinity (Hong et al., 2001Hong S. Choi G. Park S. Chung A.S. Hunter E. Rhee S.S. Type D retrovirus Gag polyprotein interacts with the cytosolic chaperonin TRiC.J. Virol. 2001; 75: 2526-2534Crossref PubMed Scopus (51) Google Scholar; data not shown). p4 addition affected the same residues in ApiCCT3 as p6 did (data not shown), albeit to a lower extent. In contrast, no perturbations were observed upon addition of Box1 (data not shown).Figure S2Specific Amino Acid Labeling and 19F Strategies Guide NMR Spectral Assignment of ApiCCT3, Related to Figure 2Show full caption(A) Overlay of specific amino acid-labeled ApiCCT3 spectra onto a uniformly 15N labeled ApiCCT3 spectra (gray). 15N His, 15N Met, 15N Arg, 15N Lys, and 15N Gly are colored green, purple, cyan, blue, and red, respectively.(B) Schematic summarizing the assigned amino acid positions for the panel of 15N-specific amino acid-labeling schemes colored as in Figure S2A.(C and D) 15N specific amino acid labeled together with mutagenesis of individual residues were used to identify unambiguously histidine (C) and methionine (D) peaks, these positions were used as anchors in the backbone assignment process. The ApiCCT3 protein is colored white and is shown in cartoon representation. The WT ApiCCT3 spectra are shown in red and are overlaid with spectra obtained with 15N His or 15N Met labeled ApiCCT3 domains carrying alanine mutations at respective methionine and histidine positions.(E) 19F tyrosine-labeled ApiCCT3 proteins were purified and spectra obtained for each tyrosine to phenylalanine substitution to facilitate the assignment of each peak in the spectrum. ApiCCT3 spectra shown from top to bottom: Y359F, Y303F, Y274F, Y247F, Y232F, and WT.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Overlay of specific amino acid-labeled ApiCCT3 spectra onto a uniformly 15N labeled ApiCCT3 spectra (gray). 15N His, 15N Met, 15N Arg, 15N Lys, and 15N Gly are colored green, purple, cyan, blue, and red, respectively. (B) Schematic summarizing the assigned amino acid positions for the panel of 15N-specific amino acid-labeling schemes colored as in Figure S2A. (C and D) 15N specific amino acid labeled together with mutagenesis of individual residues were used to identify unambiguously histidine (C) and methionine (D) peaks, these positions were used as anchors in the backbone assignment process. The ApiCCT3 protein is colored white and is shown in cartoon representation. The WT ApiCCT3 spectra are shown in red and are overlaid with spectra obtained with 15N His or 15N Met labeled ApiCCT3 domains carrying alanine mutations at respective methionine and histidine positions. (E) 19F tyrosine-labeled ApiCCT3 proteins were purified and spectra obtained for each tyrosine to phenylalanine substitution to facilitate the assignment of each peak in the spectrum. ApiCCT3 spectra shown from top to bottom: Y359F, Y303F, Y274F, Y247F, Y232F, and WT. Given that Y247 in ApiCCT3 (Figures 2A and 2B) was strongly perturbed upon substrate binding, we used 19F-NMR on 3F-tyrosine-labeled ApiCCT3 for an orthogonal assessment of the binding interface (Figures 2C and S2E). 1D 19F-NMR spectra of 3F-tyrosine-labeled ApiCCT3 revealed five discrete peaks, consistent with the five tyrosine residues in ApiCCT3 (Figure 2C). Systematic tyrosine-to-phenylalanine point mutations assigned each peak to unique tyrosine residues (Figure S2E). Upon addition of p6, one of the peaks exhibited a well-defined 0.2 ppm shift. In good agreement with our chemical-shift mapping, the perturbed peak corresponded to the 19F-tyrosine peak of Y247 (Figure 2C). Guided by NMR-CS information (Figure 2Di), we used CS-Rosetta and modeling to gain a structural understanding of ApiCCT3 in the substrate-bound conformation (Shen et al., 2009Shen Y. Vernon R. Baker D. Bax A. De novo protein structure generation from incomplete chemical shift assignments.J. Biomol. NMR. 2009; 43: 63-78Crossref PubMed Scopus (198) Google Scholar) (see Experimental Procedures and Figures S4D and S4E). The lowest energy models were comparable to the deposited ApiCCT3 structure without substrate (Pappenberger et al., 2002Pappenberger G. Wilsher J.A. Roe S.M. Counsell D.J. Willison K.R. Pearl L.H. Crystal structure of the CCTgamma apical domain: implications for substrate binding to the eukaryotic cytosolic chaperonin.J. Mol. Biol. 2002; 318: 1367-1379Crossref PubMed Scopus (65) Google Scholar). Of note, our NMR-derived structural model resolved the apical protrusion, not resolved in the ApiCCT3 crystal structure and shown to be intrinsically disordered in a previous NMR study of an archaeal apical domain obtained without substrate (Heller et al., 2004Heller M. John M. Coles M. Bosch G. Baumeister W. Kessler H. NMR studies on the substrate-binding domains of the thermosome: structural plasticity in the protrusion region.J. Mol. Biol. 2004; 336: 717-729Crossref PubMed Scopus (15) Google Scholar).Figure S4Structural Interpretation of p6 Dynamics using CS-Rosetta, Related to Figure 4Show full caption(A) Assigned 15N-1H spectrum for the p6 peptide. Residue numbers are shown next to the assigned peaks. Peaks denoted with gray numbers were not assigned.(B) Scatter plots illustrating the generation of low-scoring models using CS-Rosetta guided by chemical shift information for p6 using experimentally derived backbone assignments.(C) As in (B) but using backbone assignments from samples collected in 50% TFE (Fossen et al., 2005Fossen T. Wray V. Bruns K. Rachmat J. Henklein P. Tessmer U. Maczurek A. Klinger P. Schubert U. Solution structure of the human immunodeficiency virus type 1 p6 protein.J. Biol. Chem. 2005; 280: 42515-42527Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The 50% TFE structure was used as a reference.(D) 2D 1H-15N spectrum of p6 colored according to increasing concentrations of added unlabeled TRiC complex: blue is no addition, and purple is 4-fold TRiC excess. Experiments at a higher TRiC:p6 ratio resulted in near complete broadening of the 15N p6 peaks and thus was not included in this overlay.(E) CD experiments comparing changes in secondary structure between sum of components (ApiCCT3+p6, orange) and complex (ApiCCT3:p6, blue), inset monitoring helical content at 222 nm for two different chaperone/peptide ratios. At 1:1 (ApiCCT3:p6) ratios we observe a minimal change in helical signal, even when the equilibrium is shifted entirely to complex formation at 2:1 (ApiCCT3:p6) ratios we still observe very small decreases in helical content.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Assigned 15N-1H spectrum for the p6 peptide. Residue numbers are shown next to the assigned peaks. Peaks denoted with gray numbers were not assigned. (B) Scatter plots illustrating the generation of low-scoring models using CS-Rosetta guided by chemical shift information for p6 using experimentally derived backbone assignments. (C) As in (B) but using backbone assignments from samples collected in 50% TFE (Fossen et al., 2005Fossen T. Wray V. Bruns K. Rachmat J. Henklein P. Tessmer U. Maczurek A. Klinger P. Schubert U. Solution structure of the human immunodeficiency virus type 1 p6 protein.J. Biol. Chem. 2005; 280: 42515-42527Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The 50% TFE structure was used as a reference. (D) 2D 1H-15N spectrum of p6 colored according to increasing concentrations of added unlabeled TRiC complex: blue is no addition, and purple is 4-fold TRiC excess. Experiments at a higher TRiC:p6 ratio resulted in near complete broadening of the 15N p6 peaks and thus was not included in this overlay. (E) CD experiments comparing changes in secondary structure between sum of components (ApiCCT3+p6, orange) and complex (ApiCCT3:p6, blue), inset monitoring helical content at 222 nm for two different chaperone/peptide ratios. At 1:1 (ApiCCT3:p6) ratios we observe a minimal change in helical signal, even when the equilibrium is shift" @default.
• W2010966202 created "2016-06-24" @default.
• W2010966202 creator A5041204065 @default.
• W2010966202 creator A5053361384 @default.
• W2010966202 creator A5069731625 @default.
• W2010966202 creator A5077594305 @default.
• W2010966202 creator A5079218743 @default.
• W2010966202 date "2014-11-01" @default.
• W2010966202 modified "2023-10-13" @default.
• W2010966202 title "The Structural Basis of Substrate Recognition by the Eukaryotic Chaperonin TRiC/CCT" @default.
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