Abstract
Molecular chaperones act on non-native proteins in the cell to prevent their aggregation, premature folding or misfolding. Different chaperones often exert distinct effects, such as acceleration or delay of folding, on client proteins via mechanisms that are poorly understood. Here we report the solution structure of SecB, a chaperone that exhibits strong antifolding activity, in complex with alkaline phosphatase and maltose-binding protein captured in their unfolded states. SecB uses long hydrophobic grooves that run around its disk-like shape to recognize and bind to multiple hydrophobic segments across the length of non-native proteins. The multivalent binding mode results in proteins wrapping around SecB. This unique complex architecture alters the kinetics of protein binding to SecB and confers strong antifolding activity on the chaperone. The data show how the different architectures of chaperones result in distinct binding modes with non-native proteins that ultimately define the activity of the chaperone.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Bukau, B., Weissman, J. & Horwich, A. Molecular chaperones and protein quality control. Cell 125, 443–451 (2006)
Kim, Y. E., Hipp, M. S., Bracher, A., Hayer-Hartl, M. & Hartl, F. U. Molecular chaperone functions in protein folding and proteostasis. Annu. Rev. Biochem. 82, 323–355 (2013)
Saibil, H. Chaperone machines for protein folding, unfolding and disaggregation. Nature Rev. Mol. Cell Biol. 14, 630–642 (2013)
Horwich, A. L. Molecular chaperones in cellular protein folding: the birth of a field. Cell 157, 285–288 (2014)
Mattoo, R. U. H. & Goloubinoff, P. Molecular chaperones are nanomachines that catalytically unfold misfolded and alternatively folded proteins. Cell. Mol. Life Sci. 71, 3311–3325 (2014)
Sala, A., Bordes, P. & Genevaux, P. Multitasking SecB chaperones in bacteria. Front. Microbiol. 5, 666 (2014)
Hartl, F. U., Lecker, S., Schiebel, E., Hendrick, J. P. & Wickner, W. The binding cascade of SecB to SecA to SecY/E mediates preprotein targeting to the E. coli plasma membrane. Cell 63, 269–279 (1990)
Hardy, S. J. & Randall, L. L. A kinetic partitioning model of selective binding of nonnative proteins by the bacterial chaperone SecB. Science 251, 439–443 (1991)
Wolff, N., Sapriel, G., Bodenreider, C., Chaffotte, A. & Delepelaire, P. Antifolding activity of the SecB chaperone is essential for secretion of HasA, a quickly folding ABC pathway substrate. J. Biol. Chem. 278, 38247–38253 (2003)
Bechtluft, P. et al. Direct observation of chaperone-induced changes in a protein folding pathway. Science 318, 1458–1461 (2007)
Zhou, J. & Xu, Z. Structural determinants of SecB recognition by SecA in bacterial protein translocation. Nature Struct. Biol. 10, 942–947 (2003)
Ullers, R. S. et al. SecB is a bona fide generalized chaperone in Escherichia coli. Proc. Natl Acad. Sci. USA 101, 7583–7588 (2004)
Knoblauch, N. T. et al. Substrate specificity of the SecB chaperone. J. Biol. Chem. 274, 34219–34225 (1999)
Ullers, R. S., Ang, D., Schwager, F., Georgopoulos, C. & Genevaux, P. Trigger factor can antagonize both SecB and DnaK/DnaJ chaperone functions in Escherichia coli. Proc. Natl Acad. Sci. USA 104, 3101–3106 (2007)
Sakr, S. et al. Lon protease quality control of presecretory proteins in Escherichia coli and its dependence on the SecB and DnaJ (Hsp40) chaperones. J. Biol. Chem. 285, 23506–23514 (2010)
Calloni, G. et al. DnaK functions as a central hub in the E. coli chaperone network. Cell Reports 1, 251–264 (2012)
Castanié-Cornet, M.-P., Bruel, N. & Genevaux, P. Chaperone networking facilitates protein targeting to the bacterial cytoplasmic membrane. Biochim. Biophys. Acta 1843, 1442–1456 (2014)
Baars, L. et al. Defining the role of the Escherichia coli chaperone SecB using comparative proteomics. J. Biol. Chem. 281, 10024–10034 (2006)
Tang, Y., Pan, X., Tai, P. C. & Sui, S.-F. The structure of SecB/OmpA as visualized by electron microscopy: The mature region of the precursor protein binds asymmetrically to SecB. Biochem. Biophys. Res. Commun. 393, 698–702 (2010)
Saio, T., Guan, X., Rossi, P., Economou, A. & Kalodimos, C. G. Structural basis for protein antiaggregation activity of the trigger factor chaperone. Science 344, 1250494 (2014)
Rosenzweig, R., Moradi, S., Zarrine-Afsar, A., Glover, J. R. & Kay, L. E. Unraveling the mechanism of protein disaggregation through a ClpB-DnaK interaction. Science 339, 1080–1083 (2013)
Zhuravleva, A., Clérico, E. M. & Gierasch, L. M. An interdomain energetic tug-of-war creates the allosterically active state in Hsp70 molecular chaperones. Cell 151, 1296–1307 (2012)
Kerfah, R., Plevin, M. J., Sounier, R., Gans, P. & Boisbouvier, J. Methyl-specific isotopic labeling: a molecular tool box for solution NMR studies of large proteins. Curr. Opin. Struct. Biol. 32, 113–122 (2015)
Murén, E. M., Suciu, D., Topping, T. B., Kumamoto, C. A. & Randall, L. L. Mutational alterations in the homotetrameric chaperone SecB that implicate the structure as dimer of dimers. J. Biol. Chem. 274, 19397–19402 (1999)
Xu, Z., Knafels, J. D. & Yoshino, K. Crystal structure of the bacterial protein export chaperone secB. Nature Struct. Biol. 7, 1172–1177 (2000)
Dekker, C., de Kruijff, B. & Gros, P. Crystal structure of SecB from Escherichia coli. J. Struct. Biol. 144, 313–319 (2003)
Burmann, B. M., Wang, C. & Hiller, S. Conformation and dynamics of the periplasmic membrane-protein–chaperone complexes OmpX–Skp and tOmpA–Skp. Nature Struct. Mol. Biol. 20, 1265–1272 (2013)
Krishnamurthy, V. M., Semetey, V., Bracher, P. J., Shen, N. & Whitesides, G. M. Dependence of effective molarity on linker length for an intramolecular protein-ligand system. J. Am. Chem. Soc. 129, 1312–1320 (2007)
Gelis, I. et al. Structural basis for signal-sequence recognition by the translocase motor SecA as determined by NMR. Cell 131, 756–769 (2007)
Clare, D. K., Bakkes, P. J., van Heerikhuizen, H., van der Vies, S. M. & Saibil, H. R. Chaperonin complex with a newly folded protein encapsulated in the folding chamber. Nature 457, 107–110 (2009)
Popovych, N., Tzeng, S. R., Tonelli, M., Ebright, R. H. & Kalodimos, C. G. Structural basis for cAMP-mediated allosteric control of the catabolite activator protein. Proc. Natl Acad. Sci. USA 106, 6927–6932 (2009)
Milbradt, A. G. et al. Increased resolution of aromatic cross peaks using alternate 13C labeling and TROSY. J. Biomol. NMR 62, 291–301 (2015)
Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995)
Güntert, P. Automated NMR structure calculation with CYANA. Methods Mol. Biol. 278, 353–378 (2004)
Shen, Y., Delaglio, F., Cornilescu, G. & Bax, A. TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. J. Biomol. NMR 44, 213–223 (2009)
Brunger, A. T. Version 1.2 of the Crystallography and NMR system. Nature Protocols 2, 2728–2733 (2007)
Chun, S. Y., Strobel, S., Bassford, P., Jr & Randall, L. L. Folding of maltose-binding protein. Evidence for the identity of the rate-determining step in vivo and in vitro. J. Biol. Chem. 268, 20855–20862 (1993)
Betton, J. M., Sassoon, N., Hofnung, M. & Laurent, M. Degradation versus aggregation of misfolded maltose-binding protein in the periplasm of Escherichia coli. J. Biol. Chem. 273, 8897–8902 (1998)
Marsh, J. A., Singh, V. K., Jia, Z. & Forman-Kay, J. D. Sensitivity of secondary structure propensities to sequence differences between α- and γ-synuclein: implications for fibrillation. Protein Sci. 15, 2795–2804 (2006)
Camilloni, C., De Simone, A., Vranken, W. F. & Vendruscolo, M. Determination of secondary structure populations in disordered states of proteins using nuclear magnetic resonance chemical shifts. Biochemistry 51, 2224–2231 (2012)
Betton, J. M. & Hofnung, M. Folding of a mutant maltose-binding protein of Escherichia coli which forms inclusion bodies. J. Biol. Chem. 271, 8046–8052 (1996)
Tejero, R., Snyder, D., Mao, B., Aramini, J. M. & Montelione, G. T. PDBStat: a universal restraint converter and restraint analysis software package for protein NMR. J. Biomol. NMR 56, 337–351 (2013)
Acknowledgements
The work was supported by National Institutes of Health grant GM073854 to C.G.K.
Author information
Authors and Affiliations
Contributions
C.H. and C.G.K. designed the project. C.H. performed protein purification, NMR data collection and analysis, structure determination, and kinetic and thermodynamic assays. P.R. assisted with structure determination. T.S. assisted with NMR analysis and kinetic assays. C.H. and C.G.K. wrote the manuscript.
Corresponding author
Additional information
Reviewer Information Nature thanks L. Kay and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
Extended Data Figure 1 NMR characterization of SecB and unfolded MBP.
a, SecB is enriched in hydrophobic amino acids, such as methyl-bearing (Ala, Ile, Leu, Met, Thr and Val) and aromatic (Phe and Tyr). b, 1H–15N TROSY HSQC (left) and 1H–13C methyl HMQC (right) spectra of [U-2H; Ala-13CH3; Met-13CH3; Ile-δ1-13CH3; Leu,Val-13CH3/13CH3; Thr-13CH3]-labelled SecB. SecB packing gives rise to two pairs of spectroscopically equivalent subunits: one pair is formed by subunits A and D, and the other pair by subunits B and C. Select assignment is included in the methyl spectrum with the asterisk indicating the other pair. c, 1H–15N HSQC spectra of select MBP fragments spanning the entire sequence of MBP. d, Secondary structure propensity (SSP) values39,40 of unfolded MBP (extracted collectively from the fragments) plotted as a function of the amino-acid sequence. A SSP score at a given residue of 1 or −1 reflects a fully formed α-helical or β-structure (extended), respectively, whereas a score of, for example, 0.5 indicates that 50% of the conformers in the native-state ensemble of the protein are helical at that position. The data show that several of the secondary structure elements in the folded MBP retain some transient secondary structure in the unfolded MBP fragments.
Extended Data Figure 2 NMR characterization of PhoA and MBP binding to SecB.
a, To determine the SecB-recognition sites within PhoA and MBP, 15N-labelled PhoA and MBP fragments were titrated with unlabelled SecB. Owing to the labelling scheme and the size of SecB, the intensity of the PhoA and MBP residues that are bound by SecB decreases dramatically or disappears. Several titration points were recorded but here only the spectra for the SecB:PhoA and SecB:MBP 1:1 are shown for two select fragments. The 1H–15N HSQC spectra of PhoA or MBP are shown in the absence (blue) and presence (red) of SecB. b, c, PhoA (b) and MBP (c) refolding in the presence and absence of SecB monitored by 1H–15N HSQC spectra. Spectra of the ‘refolded’ state were recorded after rapid dilution of urea-treated MBP/PhoA in native buffer. Spectra of the ‘unfolded’ state were recorded in urea. MBP and PhoA refolded in their native structure in the absence of SecB but were retained in the unfolded state in the presence of SecB.
Extended Data Figure 3 Energetics of SecB interaction with PhoA and MBP.
a, MALS of SecB−PhoA complex showing a stoichiometry of 1:1. b, ITC of SecB binding to PhoA and the energetics of binding. c, Kd values for complexes between select PhoA fragments encompassing the five (a–e) SecB-recognition sites and SecB. d, MALS of SecB−MBP complex showing a stoichiometry of 1:1. e, ITC of SecB binding to MBP and the energetics of binding. f, Kd values for complexes between select MBP fragments encompassing the seven (a–g) SecB-recognition sites and SecB. More than one of the smaller PhoA or MBP fragments (for example, PhoAc, PhoAd–e, MBPc–d) can be accommodated within SecB. Of note is the large favourable enthalpy of binding for the interaction of MBP and PhoA with SecB reflecting the large interacting surface. However, a large but unfavourable entropy diminishes the overall binding.
Extended Data Figure 4 NMR characterization of the SecB−PhoA complex.
a, 1H–15N TROSY HSQC spectra of PhoA in the unfolded state (light blue) and in complex with SecB (grey). The unfolded state was induced by the addition of reducing agent20 or urea and assigned and characterized by NMR as shown before20. Select resonance assignment of SecB-recognition sites in PhoA is included (the colour is per the colour code for each SecB-recognition site within PhoA; see Fig. 1b). There is an excellent correspondence between the PhoA residues identified to bind to SecB using the various PhoA fragments (Extended Data Fig. 2a) and the residues of full-length PhoA that are bound to SecB in the SecB−PhoA complex. All five SecB-recognition sites in PhoA (a–e) are engaged by SecB in the SecB−PhoA complex. The PhoA regions that are not bound to SecB (they retain their intensity in the complex) are all in an unfolded conformation as suggested by their essentially identical chemical shifts to the unfolded PhoA. b, Select strips from 13C-edited NOESY experiments highlighting intermolecular NOEs in the SecB−PhoA complex. Owing to severe resonance overlap in the 120 kDa SecB−PhoA complex, to identify specific intermolecular NOEs we prepared samples wherein the two protein partners are labelled in different methyl-bearing type of amino acids. In this example, SecB was labelled in Leu, Met and Val residues and PhoA in Ile residues. Thus, all NOEs detected between Leu/Val/Met and Ile methyls are intermolecular. c, 1H–13C methyl HMQC spectra of SecB in complex with PhoA fragments carrying the individual PhoA sites: PhoAa (green), PhoAc (orange), PhoAd (magenta) and PhoAe (red). Both SecB and PhoA fragments are [U-2H; Ala-13CH3; Met-13CH3; Ile-δ1-13CH3; Leu,Val-13CH3/13CH3; Thr-13CH3]-labelled. d, Representative strips from 13C-edited NOESY–HSQC and HMQC–NOESY–HMQC NMR experiments. The NOE cross-peaks between SecB and residues of PhoA fragments are designated by a dashed-line red circle. e, Characteristic NOEs showing that the primary binding groove in SecA is enlarged by the displacement of helix α2 as shown in Fig. 4a. For example, the NOE between SecB residues Ala95 and Phe137 is consistent with the closed conformation observed in apo SecB. This NOE is not present in the SecB−PhoA complex because the two SecB residues have moved apart as a result of the displacement of the helix α2.
Extended Data Figure 5 Strategy for the structure determination of the SecB−PhoA complex.
The three main steps are briefly described here. More details can be found in Methods. The lowest-energy NMR structures of the SecB complexes with the individual PhoA sites a, c, d and e are shown. The structural and NMR statistics for each structure are shown in Extended Data Table 1 and Methods.
Extended Data Figure 6 Structures of SecB with MBP sites.
a, Lowest-energy structure of SecB in complex with a MBP fragment encompassing site d (MBPd, residues 105–152). b, Lowest-energy structure of SecB in complex with a MBP fragment encompassing site e (MBPe, residues 165–210). SecB is shown as grey solvent-accessible surface (left) or as white cartoon (right). Expanded views (right) of the contacts between SecB and MBP. The SecB residues mediating contacts with MBP are shown as blue ball-and-stick. In both complexes an additional MBP molecule binds symmetrically to the opposite face of SecB but are not shown for clarity.
Extended Data Figure 7 NMR-driven model structure of SecB−MBP complex.
a, 1H–15N TROSY HSQC spectra of MBP fragments (grey), MBP fragments in complex with SecB (blue) and full-length MBP in complex with SecB (magenta). The Gly (left) and Trp Nε (right) regions are shown as examples because of the excellent dispersion and lack of severe resonance overlap. The various MBP fragments covering the entire MBP sequence (Extended Data Fig. 1c) are coloured grey and if they are located within a SecB-recognition site it is denoted in the superscript. The MBP residues that do not interact with SecB retain their intensity. These are residues located in regions that are not SecB-recognition sites (Fig. 1c). When these spectra are compared with the spectra of full-length MBP in complex with SecB (in magenta) a very good resonance correspondence is observed. Thus, two important observations can be made: first, all seven SecB-recognition sites (a–g) in MBP are engaged by SecB in the SecB−MBP complex; and, second, the MBP regions that do not interact with SecB in the SecB−MBP complex remain in an unfolded state. The Trp spectra (right) provide direct evidence in support of these observations: all Trp residues, with the exception of Trp155, are located in SecB-recognition sites and they all interact with SecB in the SecB−MBP complex. In contrast, Trp155 does not bind to SecB when the corresponding MBP fragment was used, and this was also the case for MBP. b, Modelled structure of the SecB−MBP complex. SecB is shown as a solvent-exposed surface and MBP as a pink ribbon. The seven MBP sites recognized by SecB are shown as side-chain surface and coloured per the colour code in the graphic of the MBP sequence at the top. The structure of the complex was modelled as detailed in Methods. Briefly, as mentioned above, NMR analysis demonstrated that all seven recognition sites in MBP (labelled a–g) are bound to SecB in the SecB−MBP complex. We have determined the high-resolution structure of MBPd and MBPe in complex with SecB (Extended Data Fig. 6). Because of their length and the short linker tethering the two sites, d and e, most probably bind to the same side of SecB. MBP site f is the longest one, consisting of ~90 residues, and is thus entirely accommodated on the other side of SecB. With sites d, e and f occupying the primary binding sites, the other recognition sites (a, b, c and g), being much shorter, can be accommodated within the secondary client-binding sites on SecB. The structure of MBP sites d and e in complex with SecB was determined using the experimental intermolecular NOE data. The hydrophobic residues of the sites a, b, c, f and g showing the strongest effect upon SecB binding, as determined by differential line broadening, were used to drive the docking of these sites to non-polar residues on SecB. The modelled structure shows that the entire MBP sequence can be accommodated within one SecB molecule.
Extended Data Figure 8 Anti-aggregation activity of SecB.
a, A triple amino-acid substitution in the SecB (V40A/L42A/L44A) client-binding site was prepared and is referred to as the triple mutant SecB (SecBTM). ITC profile of the binding of PhoA to SecBTM to be compared with PhoA binding to wild-type SecB (Extended Data Fig. 3b). The triple substitution causes a 40-fold reduction in the affinity of SecB for PhoA. b, Fluorescence-monitored MBP folding in the absence of SecB (blue), in the presence of wild-type SecB (green) and in the presence of SecBTM (red). The triple mutant diminishes significantly the antifolding activity of SecB. c, 1H–15N TROSY HSQC spectra of MBP refolded in the absence (blue) and presence of SecBTM (red). In contrast to wild-type SecB (Extended Data Fig. 2c), SecBTM cannot hold MBP in the unfolded state. d, 1H–13C methyl HMQC spectra of MBPmut (blue) and in the presence of SecB (red) recorded at 22 °C. The MBP mutant (MBPmut) carries two amino-acid substitutions (G32D/I33P) that renders the protein prone to aggregation41, especially at temperatures above 30 °C. No NMR signal of MBPmut can be detected at temperatures above 30 °C and the protein precipitates in the NMR tube. At 22 °C, MBPmut is folded, as evidenced by the resonance dispersion in the NMR spectra, and does not interact with SecB. e, 1H–13C methyl HMQC spectrum of MBPmut in the presence of SecB recorded at 50 °C. MBPmut suffers heavy precipitation and aggregation at temperatures higher than 30 °C, but in the presence of SecB it is stable and folded even at temperatures as high as 50 °C. f, 1H–15N TROSY HSQC spectra of SecB (blue) and in the presence of MBPmut (orange) at 42 °C, indicating binding. Because of the elevated temperature, a significant unfolded population of MBPmut is present, which binds to SecB (see main text). g, Mapping of the sites (orange) used by SecB to interact with MBPmut, on the basis of the chemical shift perturbation data from the spectra in f.
Extended Data Figure 9 Kinetics of PhoA and MBP interaction with SecB and TF.
a–c, SPR analysis of the interaction of SecB with PhoA (a) and MBP at 20 °C (b) and 30 °C (c). Single-cycle and multiple-cycle procedures were used for the SPR analysis of SecB with PhoA and MBP, respectively. d–f, BLI analysis of the binding of MBP to SecB (d), SecBTM (e) and TF (f). His-tagged PhoA or MBP (for SPR) or biotinylated MBP (for BLI) experiments was immobilized on an NTA chip (SPR) or streptavidin biosensor (BLI) and interactions were examined at different SecB or TF concentrations as indicated. Binding is reported in response units (RU) for SPR and wavelength shift (nanomteres) for BLI as a function of time. g, h, Effect of SecB on the kinetics of MBP folding. g, Fluorescence-monitored folding of MBP (pre form) and mature MBP (h) in the absence (blue) and presence of one- (green) and fourfold (purple) excess of SecB. SecB does not appreciably delay folding of mature MBP. In fact, SecB excess appears to increase the yield of soluble, folded mature MBP (purple). i, 1H–15N TROSY HSQC spectra of mature MBP refolded in the absence (blue) and presence of SecB (red). SecB cannot retain the mature MBP unfolded. j, Fluorescence-monitored folding of the slowly folding MBPY283D variant in the absence (blue), and presence of one- (green) and fivefold (orange) TF. As elaborated in the main text, TF does not delay folding of pre-MBP (Fig. 5a). However, it does delay folding of an inherently slowly folding MBP mutant (MBPY283D), thus highlighting the importance of the intrinsic folding of the client protein and its association rate to the chaperone.
Rights and permissions
About this article
Cite this article
Huang, C., Rossi, P., Saio, T. et al. Structural basis for the antifolding activity of a molecular chaperone. Nature 537, 202–206 (2016). https://doi.org/10.1038/nature18965
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature18965
This article is cited by
-
Dynamic interactions between E-cadherin and Ankyrin-G mediate epithelial cell polarity maintenance
Nature Communications (2023)
-
Annotating Macromolecular Complexes in the Protein Data Bank: Improving the FAIRness of Structure Data
Scientific Data (2023)
-
Bioinspired Self-assembly Nanochaperone Inhibits Tau-Derived PHF6 Peptide Aggregation in Alzheimer’s Disease
Chinese Journal of Polymer Science (2022)
-
Interactions between nascent proteins and the ribosome surface inhibit co-translational folding
Nature Chemistry (2021)
-
Mechanism of the small ATP-independent chaperone Spy is substrate specific
Nature Communications (2021)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
