Crowding Activates Heat Shock Protein 90

Abstract

Hsp90 is a dimeric ATP-dependent chaperone involved in the folding, maturation, and activation of diverse target proteins. Extensive in vitro structural analysis has led to a working model of Hsp90's ATP-driven conformational cycle. An implicit assumption is that dilute experimental conditions do not significantly perturb Hsp90 structure and function. However, Hsp90 undergoes a dramatic open/closed conformational change, which raises the possibility that this assumption may not be valid for this chaperone. Indeed, here we show that the ATPase activity of Hsp90 is highly sensitive to molecular crowding, whereas the ATPase activities of Hsp60 and Hsp70 chaperones are insensitive to crowding conditions. Polymer crowders activate Hsp90 in a non-saturable manner, with increasing efficacy at increasing concentration. Crowders exhibit a non-linear relationship between their radius of gyration and the extent to which they activate Hsp90. This experimental relationship can be qualitatively recapitulated with simple structure-based volume calculations comparing open/closed configurations of Hsp90. Thermodynamic analysis indicates that crowding activation of Hsp90 is entropically driven, which is consistent with a model in which excluded volume provides a driving force that favors the closed active state of Hsp90. Multiple Hsp90 homologs are activated by crowders, with the endoplasmic reticulum-specific Hsp90, Grp94, exhibiting the highest sensitivity. Finally, we find that crowding activation works by a different mechanism than co-chaperone activation and that these mechanisms are independent. We hypothesize that Hsp90 has a higher intrinsic activity in the cell than in vitro.

Keywords: ATPase; chaperone; conformational change; heat shock protein 90 (Hsp90); macromolecular crowding.

FIGURE 1.

Structural analysis of Hsp90 suggests susceptibility to molecular crowding. A, the HtpG closed conformation (left; modeled from 2CG9) compared with the open conformation (right; modeled from 2IOQ) highlights a difference in the volume accessible to a crowder. B, the normalized volume excluded from the probe is larger in the open state (red circles) versus the closed state (blue squares). C, the excluded volume difference between the open and closed states varies with probe size. This sensitivity to crowder size is quantified by the ΔV_crowd ratio in the open/closed states, calculated from panel B.

FIGURE 2.

PEG polymers activate HtpG. A, HtpG ATPase (black circles) increases with increasing concentration of PEG 20,000 (R_g of 66 Å). The dashed line illustrates the upward-curving relationship between hydrolysis rate and crowder concentration. Both DnaK (blue diamonds) and GroEL (orange triangles) are not activated by PEG. B, crowding activation of HtpG depends strongly on PEG molecular weight. A slice through the PEG concentration series at 100 mg/ml (gray dashed line) shows increasing activation with increasing polymer size. The PEG activation is fully inhibited by 20 μm radicicol (rad). C, ATPase values at 100 mg/ml PEG increase as the R_g of the crowder approaches 25 Å, but show only modest changes above that size. Buffer conditions were: 25 mm Tris, pH 9.0, 50 mm KCl, 5 mm MgCl₂, 2 mm ATP, 25 °C. Error bars indicate the S.E. for at least three measurements.

FIGURE 3.

A, PEG activation of HtpG does not correlate with PEG increases in osmotic pressure. cP, centipoise. B, cosolvents and osmolytes do not show crowding-like activation of HtpG. C, ATPase values with dextran polymers at 100 mg/ml follow a similar size dependence as PEG polymers. The solid line is a visual guide. Buffer conditions were: 25 mm Tris, pH 9.0, 50 mm KCl, 5 mm MgCl₂, 2 mm ATP, 25 °C.

FIGURE 4.

A, 100 mg/ml PEG 20,000 increases the activity of multiple Hsp90 homologs. The most dramatic ATPase enhancement is observed for Grp94, increasing the rate by a factor of 15. ATPase activity for all homologs is completely inhibited by radicicol (rad). B, Grp94 displays a strong upward-curving relationship between hydrolysis rate and crowder concentration (red circles). No ATPase activity is observed in the presence of radicicol (blue triangles). The Hsp70 class chaperone BiP is not activated by PEG (black diamonds). Buffer conditions were as described under “Experimental Procedures.” Error bars indicate the S.E. for at least three measurements.

FIGURE 5.

Eyring analysis reveals entropic activation of HtpG by PEG crowders. The natural logarithm of HtpG ATPase in PEG 3350 (0 mg/ml, red circles; 60 mg/ml, blue squares; 80 mg/ml, green diamonds; 100 mg/ml, black triangles) is plotted as a function of 1/T. The linear fit indicates a single constant activation enthalpy (ΔH^‡, see “Experimental Procedures”). The fit value of ΔH^‡ is independent of PEG concentration (inset). Buffer conditions were: 25 mm Tris, pH 9.0, 50 mm KCl, 5 mm MgCl₂, 2 mm ATP. Error bars indicate the S.E. for at least three measurements.

FIGURE 6.

PEG crowders accelerate HtpG arm closure. A, FRET kinetic analysis reveals that HtpG arm closure rate increases with increasing PEG 3350 (0 mg/ml, black circles; 60 mg/ml, blue; 100 mg/ml, green; 150 mg/ml, red). Solid lines are fits to single exponential kinetics. The inset shows the open and closed states with the fluorescently labeled sites highlighted in red and blue. B, HtpG arm reopening rate is unchanged with increasing PEG 3350 (0 mg/ml, black circles; 60 mg/ml, blue; 100 mg/ml, green; 150 mg/ml, red). Solid lines are fits to single exponential kinetics. C, the PEG concentration series displays an upward-curving relationship between arm closure rate (circles) and crowder concentration. In contrast, reopening rates (squares) are PEG-independent. Error bars indicate the S.E. for at least three measurements. D, FRET observations are consistent with an Hsp90 energy landscape wherein crowding destabilizes the open conformation, thereby increasing closure rate and leaving reopening rate unchanged.

FIGURE 7.

A, Hsp82 is activated by increasing concentrations of the aha1 co-chaperone (red circles). The addition of 60 mg/ml PEG 3350 increases the maximal activity and effective binding constant (blue squares). Solid lines are fits to the Michaelis-Menten equation. B, an energy landscape interpretation in which crowding destabilization of the open configuration and aha1 stabilization of the transition state can act in an additive manner. Buffer conditions were: 25 mm Tris, pH 9.0, 12 mm KCl, 5 mm MgCl₂, 2 mm ATP, 25 °C. Error bars indicate the S.E. for at least three measurements.