Challenges in determining the structures of heterogeneous and dynamic protein complexes have greatly hampered past efforts to obtain a mechanistic understanding of many important biological processes. One such process is chaperone-assisted protein folding. Obtaining structural ensembles of chaperone–substrate complexes would ultimately reveal how chaperones help proteins fold into their native state. To address this problem, we devised a new structural biology approach based on X-ray crystallography, termed residual electron and anomalous density (READ). READ enabled us to visualize even sparsely populated conformations of the substrate protein immunity protein 7 (Im7) in complex with the Escherichia coli chaperone Spy, and to capture a series of snapshots depicting the various folding states of Im7 bound to Spy. The ensemble shows that Spy-associated Im7 samples conformations ranging from unfolded to partially folded to native-like states and reveals how a substrate can explore its folding landscape while being bound to a chaperone.
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The authors would like to thank J. Smith, D. Akey, U. Jakob, D. Smith, Z. Wawrzak, and F. Stull for critical comments and suggestions. Use of the Advanced Photon Source, an Office of Science User Facility operated for the US Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the US DOE under contract no. DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (grant 085P1000817). This work was funded by an NRSA National Institutes of Health (NIH) grant GM108298 (L.S.A.), a Boehringer Ingelheim Fonds fellowship (P.K.), a National Natural Science Foundation of China (NSFC) grant 31400664 (S.Q.), the Shanghai Pujiang Program (S.Q.), NIH grant GM102829 (J.C.A.B.), NIH grant GM107233 (C.L.B.), NIH grant 1P01 GM063210 (P.V.), the Phenix Industrial Consortium and the US Department of Energy Contract No. DE-AC02-05CH11231 (P.V.) and NSF grant CHE1506273 (C.L.B.). J.C.A.B. is supported as a Howard Hughes Medical Institute Investigator.
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Integrated supplementary information
Supplementary Figure 1 Truncating Spy’s unstructured tails does not dramatically decrease chaperone activity in vivo or in vitro.
(a) Overexpression of Spy increases the steady state level of the unstable Im7 mutant (Im7 L53A I54A) detected via SDS-PAGE and Coomassie stain (above) or by anti-His tag antibody (below). (b) Truncating Spy’s unstructured tails does not dramatically alter chaperone activity in vitro, as determined by the aldolase refolding assay. In vitro, Spy is able to provide minor aid to aldolase refolding capacity, compared to the major folding aid provided to Im7 in vivo. Error bars plotted are s.e.m. using 3-5 replicates from separate experiments.
Substrates are Im76–45 (a), casein 148-177 (b), and Im7 L18A L19A L37A (c), respectively. Lanes W1-W5: washes of crystals in crystallization solution; lane D: dissolved crystals. The lack of protein in (b) for W2 relative to W3 for casein 148-177 and in (c) for Im7 L18A L19A L37A for W2 relative to W1 was due to some crystal breakage in the latter wash drops. WT Im7 crystals were too fragile to survive the washing process.
(a) Comparison of disordered Im76-45 helical propensity in simulation and experiment. Per-residue helicity profiles obtained from coarse-grained simulations (FF1-FF3) and NMR chemical shifts (Loïc Salmon, Logan S. Ahlstrom, Scott Horowitz, Alex Dickson, Charles L. Brooks III, and James C. A. Bardwell, unpublished data: Exp, black curve). The unmodified Im76-45 force field (FF1, red curve) yields an ensemble that overestimates the helicity. Applying an adjustment to the pseudo-dihedral potential (FF2, cyan curve) improves the agreement of the helicity profile with experiment. Rescaling the non-bonded interactions in addition to this adjustment results in a helicity distribution that closely matches the experimental profile (FF3, orange curve). (b) Native structure of WT Im7. Residues 6-45 are highlighted in orange.
(a) and (b) The two pseudo-crystal environments for Spy⎯ Im76-45 simulations. Use of two environments allows all multi-Spy interactions Im76-45 would encounter within the crystal. Both environments comprise four crystallographic Spy dimers (blue, red, yellow, and magenta) and a single peptide (orange).
(a) Flowchart of simulated data validation test. (b) Three target ensembles (cyan) overlaid with selected ensembles (magenta). RMSDs of target to selected ensembles: 5.7 Å (top left), 6.5 Å (middle right), and 3.7 Å (bottom left).
37% of data replaced with repeat data in 200 separate selections was used to calculate mean and standard error contact maps. Note the change in intensity scale for the standard error map of the selections. The contact profile of the ensembles from the bootstrapped selections has low error and displays a different binding profile than the initial MD pool.
(a) Binding of Spy 29-124 to Im7 L18A L19A L37A H40W measured via ITC. This Im7 mutant was subsequently used for competition assays with Im76-45 and Spy 29-124. (b) Casein 148-177 competes with Im7 6-45 for Spy 29-124 binding. Due to binding between Im76-45 and casein 148-177, as identified by analytical ultracentrifugation experiments of the two proteins (c), we were unable to accurately quantitate competition between Im76-45 and casein 148-177 (b). The dissociation constant of casein 148-177 to Spy 29-124 was determined by fluorescence (d) with a 1:1 stoichiometry of casein 148-177 to Spy 29-124 (e). Also, see Fig. 5c for competition of Im76-45 with Im7 L18A L19A L37A H40W. Error bars depict s.d. of n=3 technical replicates.
Supplementary Figures 1–8, Supplementary Tables 1–3 and Supplementary Note (PDF 2016 kb)
6.5 kev anomalous difference maps of pI-Phe substituted Im76–45 (PDF 6013 kb)
Ensemble members ordered by RMSD from least to most nativelike. Individual snapshots of the six-membered ensemble shown in Fig. 3. (MP4 4262 kb)
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Horowitz, S., Salmon, L., Koldewey, P. et al. Visualizing chaperone-assisted protein folding. Nat Struct Mol Biol 23, 691–697 (2016). https://doi.org/10.1038/nsmb.3237
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