Abstract
Chaperones assist in the folding of many proteins in the cell. Although the most well-studied chaperones use cycles of ATP binding and hydrolysis to assist in protein folding, a number of chaperones have been identified that promote folding in the absence of high-energy cofactors. Precisely how ATP-independent chaperones accomplish this feat is unclear. Here we characterized the kinetic mechanism of substrate folding by the small ATP-independent chaperone Spy from Escherichia coli. Spy rapidly associates with its substrate, immunity protein 7 (Im7), thereby eliminating Im7's potential for aggregation. Remarkably, Spy then allows Im7 to fully fold into its native state while it remains bound to the surface of the chaperone. These results establish a potentially widespread mechanism whereby ATP-independent chaperones assist in protein refolding. They also provide compelling evidence that substrate proteins can fold while being continuously bound to a chaperone.
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Acknowledgements
We thank S. Horowitz for critical reading of the manuscript. This work was funded by a Boehringer Ingelheim Fonds fellowship (P.K.) and US National Institutes of Health grant GM102829 (J.C.A.B. and S.E.R.), which also funded J.R.H. J.C.A.B. is supported as a Howard Hughes Investigator.
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F.S., P.K. and J.R.H. performed the experiments. All authors analyzed the data. F.S., S.E.R. and J.C.A.B. designed the study. F.S. wrote the manuscript, with contributions from all other authors.
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Integrated supplementary information
Supplementary Figure 1 Binding of Spy to Im7 L53A I54A (Im7I).
(Top) A two-step kinetic mechanism where Spy first binds Im7-L53AI54A followed by a conformational change within the complex (denoted by the red asterisk). The hyperbolically increasing dependence of kobs on Spy concentration when Spy binds Im7-L53AI54A (Fig. 3b, main text) is consistent with this mechanism, in which the fluorescence decrease reflects the conformational change within the complex. The plot of kobs versus Spy concentration is hyperbolic in part because kobs depends on the fractional concentration of the Im7-L53AI54A-Spy complex prior to the conformational change1,2, which changes with Spy concentration based on the Kd for the initial binding step. (Bottom) Overlay of traces for Spy binding Im7-L53AI54A at different Spy concentrations. The traces at all Spy concentrations extrapolate back to the fluorescence of Im7-L53AI54A alone at time zero, indicating that there is no burst phase and that no fluorescence change occurs with the initial binding step.
1. Vogt, A. D. & Di Cera, E. Conformational selection or induced fit? A critical appraisal of the kinetic mechanism. Biochemistry 51, 5894–5902 (2012).
2. Gianni, S., Dogan, J. & Jemth, P. Distinguishing induced fit from conformational selection. Biophys. Chem. 189, 33–39 (2014).
Supplementary Figure 2 Burst phase when Spy binds native Im7 and relative fluorescence intensity of Im7 WT–Spy complex.
(a) Burst phase size when Spy binds 4.8 µM native Im7-WT at different Spy concentrations. The initial fluorescence observable by stopped-flow when Spy binds native Im7-WT is higher than the fluorescence of Im7-WT alone (Fig. 4a, main text). The plot in a shows the fluorescence extrapolated to time zero for each trace at different Spy concentrations. The amplitude of this burst phase reaches a saturating level at high Spy concentrations, consistent with Spy binding native Im7 within the dead time of the stopped-flow instrument. The blue line is the fit to a square hyperbola, which gives a half-saturation concentration of 36 ± 11 µM. (b) A comparison of the change in tryptophan fluorescence when Spy binds native Im7-WT with the fluorescence of Im7I. The tryptophan fluorescence of Im7-WT bound to saturating concentrations of Spy (red) is higher than the fluorescence of Im7-WT alone (black). The amplitude of the post burst phase increase in fluorescence when Spy binds Im7-WT is 0.1 fluorescence units, which is ~3% of the total fluorescence intensity difference between the Im7I mimic, Im7-L53AI54A, and Im7-WT (3.5 fluorescence units), evidence that only ~3% of Im7-WT becomes unfolded to Im7I in the Im7-Spy complex. This is consistent with the change in equilibrium constant for the Im7I-Im7N step expected from the difference in affinities of Spy for Im7I and Im7N: the equilibrium constant for the unbound Im7I-Im7N step (KIN) is 319 in 0.7 M urea (Supplementary Fig. 5), which means that 99% of the Im7 is in the native state prior to binding Spy; Spy binds Im7I with ~6-fold higher affinity than Im7N, making the Im7I-Im7N equilibrium constant 53 in 0.7 M urea when bound to Spy; this means that 98% of the Im7-WT should be in the native state when bound to Spy, and that ~1% of Im7N should unfold to Im7I upon binding Spy.
Supplementary Figure 3 Im7 WT partially unfolds while it is bound to Spy.
The decreasing dependence of kobs on Spy concentration of the post-burst phase step (Fig. 3c) indicates that Spy induces the unfolding of a small proportion of the Im7N in Im7-WT into Im7I. In addition, the burst phase seen when Spy binds Im7-WT (Fig. 4a) confirms that Spy is capable of binding Im7N. These two points on their own do not address whether Im7 must be released from Spy for the partial unfolding of Im7-WT to occur (a), or if Im7 can be directly unfolded while in complex with Spy (b). (c,d) The simulated dependence of kobs on Spy concentration for the mechanisms shown in a and b, respectively. The simulated dependence of kobs on Spy concentration is shown as a blue line and the black points are the experimental kobs values from Fig. 3c, main text. The simulated dependence of kobs on Spy concentration for the mechanism omitting folding/unfolding of Im7 while bound (c) shows that kobs should reach 0 s-1 at high Spy concentrations. However, the simulated dependence of kobs on Spy concentration for the mechanism allowing folding/unfolding of Im7 while bound (d) shows that kobs should approach the sum of the folding and unfolding rate constants while bound at high Spy concentrations (in this case, 3.5 s-1). The trend in d clearly matches the trend of the observed data better than in c, suggesting that the partial unfolding of Im7-WT by binding to Spy occurs while bound to the chaperone. The difference in the half-saturation concentration between the simulated and experimental data in d is due to the mechanism c being an oversimplification of the complete mechanism, since Im7I is also in equilibrium with Im7U. If the complete mechanism (e) is used with the rate constants obtained by global analysis (Table 1 and Supplementary Fig. 7), there is excellent agreement between the observed and simulated data (f).
Supplementary Figure 4 Primary analysis of stopped-flow traces of Im7 folding in the presence of Spy.
(a) 4.8 µM Im7 folding in the absence of Spy. The trace can be fitted with a single exponential (blue line). (b) 4.8 µM Im7 folding in the presence of 5 µM Spy dimer. The trace cannot be adequately fitted with a single exponential (blue line), but can be fitted with a double exponential function (red line). (c) Kinetic amplitudes for the two phases detectable at low Spy concentrations (corresponding to kobs1 and kobs2 in g and h). The amplitude for the first phase (ΔA1) decreases with increasing Spy concentration, whereas the amplitude for the second phase (ΔA2) increases. ΔA1 decreases to the point where traces with ≥83 µM Spy can be fit with a single exponential. (d) 4.8 µM Im7 folding in the presence of 83 µM Spy dimer. The trace can be fitted with a single exponential (blue line). (e,f) 4.8 µM Im7 folding in the presence of 165 and 660 µM Spy dimer, respectively. Traces with ≥165 µM Spy showed an increase in fluorescence in the first 20 msec, which is likely the conversion of Im7U to Im7I that only becomes detectable at high Spy concentrations. As a result, transients at these concentrations had to be fitted to a double exponential function (blue line). (g) Dependence of the observed rate constant on Spy concentration for the increase in fluorescence detectable only at the highest Spy concentrations (kobs1). kobs1 decreased with Spy concentration, reaching a limit of ~110 s-1. (h) Observed rate constant for the first decreasing fluorescence phase detectable only at low Spy concentrations (kobs2). At higher Spy concentrations, the amplitude of this phase is too small to fit accurately. (i) Observed rate constant for the slow decreasing fluorescence phase detectable at all Spy concentrations (kobs3). kobs3 decreases with increasing Spy concentration, reaching a limit of 3.5 ± 0.3 s-1. kobs3 is indistinguishable from the observed rate constant for Spy binding to Im7-WT under native conditions (Fig. 3c), suggesting that both exponentials are monitoring the same step in the kinetic mechanism.
Supplementary Figure 5 Folding and unfolding data for Im7 WT in the absence of Spy.
(a) Sulphate extrapolation of Im7-WT folding and unfolding to determine the equilibrium constant for the conversion of unbound Im7U to unbound Im7I (KUI). Initially, the KUI value was poorly defined for Im7 folding/unfolding in 40 mM HEPES-KOH, pH 7.5, 100 mM NaCl, 10°C because the folding intermediate is relatively unstable under those conditions, which produces very little curvature in the folding arm of the chevron plot. To better define KUI under our standard conditions, we collected data for Im7 folding and unfolding at several concentrations of Na2SO4, which stabilizes the folding intermediate of Im7. The inset shows the sulphate dependence of KUI. Extrapolation back to 0 M Na2SO4 indicates that KUI is 1.39 ± 0.36 in 40 mM HEPES-KOH, pH 7.5, 100 mM NaCl, 10°C. (b) The denaturant dependence of Im7 folding and unfolding kinetics in 40 mM HEPES-KOH, pH 7.5, 100 mM NaCl, 10°C fitted with a KUI value of 1.39. A good fit was obtained when KUI was fixed at 1.39. The values for KUI, kIN, and kNI in 0.7 M urea (the urea concentration in the experiments used for global fitting, Supplementary Fig. 6) were calculated using the relationship kxy = kxyH2Oe (mxy/RT)[Urea] (ref. 3). Calculated in this way, the values in 0.7 M urea are KUI = 0.57 ± 0.29, kIN = 345 ± 56 s-1 and kNI = 1.08 ± 0.04 s-1.
3. Ferguson, N., Capaldi, A. P., James, R., Kleanthous, C. & Radford, S. E. Rapid folding with and without populated intermediates in the homologous four-helix proteins Im7 and Im9. J. Mol. Biol. 286, 1597–1608 (1999).
Supplementary Figure 6 Global fitting to various kinetic mechanisms.
In all 12 panels (a-l), top: mechanism used in globally fitting the two following plots; middle: global fitting of Spy binding Im7-WT traces; bottom: global fitting of Im7 folding in the presence of Spy traces. The Spy concentrations range from 0 µM (black trace) to 660 µM (light blue trace). The black lines in the plots are the best fit to the data. Although the mechanisms in h and k superficially appear to fit the data, they fail to account for the initial increase in fluorescence in the first 20 ms of the traces for Im7 folding in the presence of the highest Spy concentrations (light blue trace). Only the kinetic mechanism that allows complete folding of Im7 while bound to Spy (l) can fit the data for Spy binding Im7-WT as well as the initial fluorescence increase and the subsequent fluorescence decrease observed when Im7 folds in the presence of Spy.
Supplementary Figure 7 Simultaneous fitting of fluorescence transients for all data to the mechanism that allows complete folding of Im7 while it is bound to Spy.
(a) and (b), Fluorescence transients for Im7 folding and unfolding, respectively, at different urea concentrations in the absence of Spy. (c) Fluorescence transients for Im7 folding in the presence of different Spy concentrations in 0.7 M urea. (d) Fluorescence transients for Spy binding to Im7-WT at different Spy concentrations in 0.7 M urea. The black lines in all plots are the best fit to the data. The goodness of fit (Χ2/Degrees of freedom) was 1.49. Data were collected in 40 mM HEPES-KOH, pH 7.5, 100 mM NaCl at 10°C. The rate constants and fluorescence scaling factors obtained from the global analysis can be found in Table 1 of the main text and Supplementary Table 1.
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Supplementary Text and Figures
Supplementary Figures 1–7 and Supplementary Table 1 (PDF 2551 kb)
Energy plot for Im7 after stress
Energy plot for Im7 after stress. During butanol or tannin stress, the concentration of Spy in the periplasm of E. coli is upregulated 500-fold to ~2 mM10,12,13. Immediately after the protein unfolding stress is removed, the concentration of Spy would still be ~2 mM. At this concentration of Spy, the most energetically favorable state of Im7 is the Im7N–Spy complex, which can be populated directly without Im7 having to dissociate from Spy. As the Spy concentration is lowered through cell growth or degradation, unbound Im7N becomes the most energetically favorable state, and the Im7N bound to Spy is released. A frequency factor of 4.8 × 108 s−1 was used to calculate the energy of the transition states.16 (MP4 1234 kb)
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Stull, F., Koldewey, P., Humes, J. et al. Substrate protein folds while it is bound to the ATP-independent chaperone Spy. Nat Struct Mol Biol 23, 53–58 (2016). https://doi.org/10.1038/nsmb.3133
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DOI: https://doi.org/10.1038/nsmb.3133
