Substrate protein folds while it is bound to the ATP-independent chaperone Spy

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.

Figure 1

Folding of the protein Im7 in the presence and absence of the chaperone Spy. Im7 is shown as a multicolored protein that is helical in both the folding intermediate (I) and in the native folded state (N) and lacks any persistent secondary structure in the unfolded state (U). Spy is shown as a blue cradle-shaped homodimer. Spy binding and release are shown as vertical arrows.

Figure 2

ITC titrations for Spy binding to wild-type Im7 and variants. (a) Titration of 150 μM Im7-L18A L19A L37A (Im7_U) in the cell with 1.65 mM Spy dimer in the syringe. (b) Titration of 50 μM Im7-L53A I54A (Im7_I) in the cell with 550 μM Spy dimer in the syringe. (c) Titration of 230 μM Im7-WT (Im7_N) in the cell with 2.53 mM Spy dimer in the syringe. All titrations were performed in 40 mM HEPES-KOH pH 7.5, 100 mM NaCl at 10 °C. The black lines in the bottom panels are the best fit of the data to a one-site model. Values reported are the mean ± s.e. of the fit.

Figure 3

Kinetics of Spy binding to Im7_U, Im7_I, and Im7_N. Graphs show the change in tryptophan fluorescence (F) associated with Spy binding to (a) 1 μM Im7-L18A L19A L37A (Im7_U), (b) 0.5 μM Im7-L53A I54A (Im7_I), and (c) 4.8 μM Im7-WT (Im7_N). Note the logarithmic timescale. The red lines are the best fit of each data set to a single exponential. The insets show the change in observed rate constant with Spy concentration for each variant of Im7. Each data point in the insets represents the average of 10-15 traces. For Spy binding to Im7-L18A L19A L37A, k_obs increased linearly with Spy concentration, indicating that Spy can bind Im7_U, and giving k_on of 1.3 ± 0.1 × 10⁷ M⁻¹s⁻¹ and k_off of 210 ± 20 s⁻¹. For Spy binding to Im7-L53A I54A, k_obs increased hyperbolically with Spy concentration, reaching a limiting value of 62 ± 2 s⁻¹, which is consistent with Spy binding Im7_I followed by partial folding or unfolding of Im7 within the complex. For Spy binding to Im7-WT, k_obs decreased with Spy concentration, reaching a limiting value of 3.3 ± 0.9 s⁻¹. Values reported are the mean ± s.e. of the fit. .In combination with the burst phase when Spy binds Im7-WT (Fig. 4a), this decreasing k_obs suggests that Spy can bind Im7_N followed by partial unfolding to Im7_I while bound.

Figure 4

Stopped-flow fluorescence traces of Spy binding Im7-WT and Im7 folding in the presence of Spy. (a) Traces for Spy binding to Im7-WT under native conditions. The fluorescence of the first data point for each trace is higher than the fluorescence of Im7-WT alone (black trace) and reaches a saturating fluorescence at high Spy concentrations. This burst phase is consistent with Spy binding Im7_N within the dead time of the instrument. (b) Traces for Im7-WT folding in the presence of different Spy concentrations. Im7 folding slows as the concentration of Spy is increased. An increase in fluorescence in the first 20 ms occurs in the traces at the highest Spy concentrations; this phase represents the conversion of Im7_U to Im7_I during Im7 folding that is slowed by Spy. The Spy dimer concentrations used in a and b are identical for each color. Note that the y axes are different in a and b, but that the final fluorescence at 1 s is the same in both experiments, suggesting that the same equilibrium is reached.

Figure 5

Global fitting of Im7+Spy kinetic data. (a) The global fitting was built upon the well-characterized mechanism^– for Im7 folding in the absence of Spy (black path). The experimental fluorescence traces for Spy binding to Im7-WT and Im7-WT folding in the presence of Spy (Fig. 4) were globally fit to different mechanisms containing various combinations of the steps in red. In the global fitting, the equilibrium constant for the Im7_U to Im7_I step (K_UI = k_UI/k_IU) and the forward and reverse rate constants for the Im7_I to Im7_N step were fixed to the values determined from the urea dependence of Im7 folding in the absence of Spy (Supplementary Fig. 5). (b, c) Attempted global fitting to the mechanism that omits and allows, respectively, the folding of Im7 while bound to Spy. For clarity, only the traces for Im7 folding in the presence of Spy are shown. The black lines in the plots are the best fit to the data. The mechanism that completely omits folding of Im7 while bound to Spy (b) fails to fit the data, whereas the mechanism that allows folding of Im7 while bound (c) can successfully fit the data. Global fitting to additional mechanisms and the best fit for the Spy binding Im7-WT data can be found in Supplementary Fig. 6.

Figure 6

Proposed mechanism for how Spy inhibits aggregation and assists protein folding in vivo. Clockwise from the bottom left (native substrate protein), exposure to chemical stress (denoted by the pink box) causes proteins in the periplasm of Escherichia coli to unfold. The stress induces the production of Spy, which rapidly associates with its substrate proteins, thereby preventing their aggregation. Once the stress is removed (denoted by the light blue box), dilution of Spy through cell growth or degradation lowers the Spy concentration, allowing the native substrate protein to be released.