It is becoming more and more apparent that structural dynamics play an important role in the function of Hsp90, and understanding the conformational ensemble is imperative for better defining its molecular mechanism. Large conformational changes occur throughout the ATPase hydrolysis cycle, and previous structural studies, as well as kinetic studies have shown that the conformational state is not rigorously determined by the bound nucleotide, but instead that nucleotide binding results in a conformational equilibrium between apo and nucleotide states.27; 29; 31
Conformational equilibria have been shown as functionally important in other systems as well. For example, NMR studies have revealed that the proto-oncogene Ras exists in a conformational equilibrium that can be shifted with point mutations. The equilibrium shift then promotes interactions with effector proteins leading to an increase in oncogenic potential.38
In another example, single molecule FRET studies revealed that even in the absence of substrate, adenylate kinase exists in both the open and closed state. Notably, the closed state, previously thought to exist only in the presence of substrate, is favored.39
These examples also support the realization that the functional pathway of Hsp90 must be considered as a stochastic process rather than as a deterministic ATP machine as has been observed for other chaperones.
Here, both SAXS and single particle EM have revealed an unanticipated solution conformation of apo HtpG that exists in a pH-dependent two-state conformational equilibrium with the previously described extended apo state. Interestingly, the apparent pKa for the conformational transition between the extended state and the Grp94-like state is 7.2 suggesting that under physiological conditions the system is optimized for maximum levels of both conformations. By using a linear combination of two structures together with rigid body modeling, we were able to dissect out the two populations of HtpG and to discover that the low pH conformation of HtpG is remarkably similar to the recent Grp94 crystal structure. While the exact rotation of the middle and N-terminal domains differs between our low pH structure and the Grp94 crystal structure, the overall architecture is surprisingly similar. Although our SAXS data is very well fit with a two state model, it is likely that a third state, the closed ATP state, is present as well. Recent single particle EM studies have directly observed the presence of the closed state under apo conditions.31
The closed state has also been identified under apo conditions for yeast by single-molecule FRET, and analysis of the energy barriers suggests that all states observed in the presence of nucleotide are accessible in the absence of nucleotide.32
If the closed state represents less than 5-10% of the total population, its effect on the SAXS modeling would be negligible. Therefore, the apo state of HtpG is most likely represented by the extended state, the Grp94-like state, and a small amount of the closed state. The conformational equilibrium of HtpG persists in the presence of saturating AMPPNP with the closed state now being significantly populated. The AMPPNP-bound equilibrium can also be shifted with pH with the Grp94-like conformation most populated at low pH and with the closed state most populated at high pH.
Given that the Grp94-like state has both conformational features of the extended (NTD-MD angle) and closed states (MD-CTD angle), it is possible that the low pH Grp94-like state (G) represents an intermediate step along the pathway proceeding from the extended model (E) to the closed model (C) (1
). Alternatively, the Grp94-like state could be off-pathway (2
) or the Grp94-
like and extended states could be combined in a three-way pathway with the closed state (3
). Typically, kinetics measurements are used to determine the connectivity of a particular pathway. However it seemed that similar information should be extractible from the pH equilibrium data. We know from the mutant HtpG SAXS data in the presence of AMPPNP that both the extended and the Gpr94-like states can convert to the closed state suggesting that pathway (3
) is the relevant pathway. Given that the only structural changes required for the transformation from the extended to the Grp94-like state occurs at the MC interface, we assumed that this pH-dependent equilibrium is unaffected by the addition of AMPPNP. With this assumption, we modeled the pathways using standard kinetic equations and confirmed that only a model allowing all three states to interconvert (3
) can recapitulate the steady-state levels observed in the presence of AMPPNP. The modeling indicates that the conversion from the extended to Grp94-like state and the extended to closed state both have a similar pKa, whereas the conversion from the Grp94-like state to the closed state is independent of pH. This suggests that the main role of pH is to shift the equilibrium for the MC domain conformation. Because the Grp94-like state is stabilized in the presence of AMPPNP, the dominant pathway from the extended state to the ATP-state most likely goes through the Grp94-like state, despite the fact that all three species can interconvert. A recent hydrogen exchange study of HtpG further supports this idea with the demonstration of an intermediate state between the `relaxed' apo state and the `tense' ATP state.30
Using the histidine mutations H446K and H446E, we probed the functional role of the two apo conformations. Using a citrate synthase aggregation assay, we demonstrate that, at least for this substrate protein, the different apo conformations of HtpG are functionally quite distinct. The Grp94-like conformation is very efficient at preventing aggregation whereas the extended conformation has little or no effect. This result was surprising, in that we anticipated the extended conformation to be better able to accommodate the large (50kD), dimeric citrate synthase. The wild type protein showed an intermediate ability to suppress aggregation as expected for being a nearly 1:1 mixture of the two conformations. Since chaperone binding via exposed hydrophobic residues on citrate synthase is likely to be the main requirement for blocking aggregation, the observed behavior suggests that the Grp94-like conformation presents hydrophobic surfaces that are better aligned to those exposed on citrate synthase. Previous work has shown that Hsp90 interacts with structured unfolding intermediates of CS,4
and that this interaction only required the MC domain of Hsp90 and was independent of nucleotide.37
This correlates well with the results presented here showing that a change in only the MC orientation of HtpG affects the interaction with CS.
One possible mechanism for the interaction is that a locally unfolded region of CS prone to aggregation could interact deep in the cleft of the Grp94-like structure binding to the hydrophobic surfaces on both MD arms of HtpG and the amphipathic α-helices in the CTD. The close juxtaposition of MD and CTD hydrophobic surfaces would not occur in the extended HtpG state. In the wild-type protein where the two conformations are in equilibrium, the citrate synthase would be able to bind to only half of the HtpG present accounting for the intermediate level of activity that is seen as compared to the two mutants. When the population is already shifted in favor of the Grp94-like structure (H446K), binding and aggregation prevention are most efficient. By contrast, the H446E mutation is unable to prevent citrate synthase aggregation because it is unable to convert to the Grp94-like state even in the presence of citrate synthase. The inability of CS to shift the conformational equilibrium of HtpG also suggests that the interaction is weak and transitory. It is also possible that the CS/HtpG interaction is mediated by local changes in HtpG that occur between the two apo states. In order to distinguish these and other possible modes of interaction, more detailed structural studies of the interaction of HtpG with CS will be required.
In order to better understand the molecular mechanism for the conformational switch, we examined the residues contacting H446 in both the extended high pH model and the Grp94-like low pH model. Unfortunately, no obvious contacts were being made that would account for the pH dependence of the conformational change. Comparison of the analogous residue to H446 in the Grp94 structure (F554) provides some insight into the molecular basis of the conformational switch. In Grp94, F554 is nicely packed in a hydrophobic pocket comprised of residues that are conserved in HtpG as hydrophobic. The one non-conserved residue contacting F554 is a serine (S725) in Grp94 and a glutamate (E604) in HtpG. This residue could explain the preference of HtpG for the Grp94-like state only when the histidine is either protonated at low pH or replaced with a lysine. Both of these would be able to form a favorable electrostatic interaction with E604 whereas the H446E mutation would be expected to clash with E604 shifting the population towards the extended state as is observed.
The use of histidines to modulate the conformation of Hsp90 appears to be a uniquely bacterial trait. The histidines involved in HtpG are not conserved in higher organisms. The similarity of the functionally active apo HtpG state to the Grp94 structure, however, suggests that Grp94 may utilize the same structural pathway as other Hsp90 homologs, and that the different structural states are conserved from bacteria to higher eukaryotes. Given the evidence that the extended, ATP and ADP states are structurally conserved from bacteria to higher eukaryotes, it is probable that the intermediate Grp94-like conformation found here is also important in higher eukaryotes. Recent FRET studies have shown that yeast Hsp90 has at least two intermediate states between the already described open apo state and closed ATP state.32; 33
One possibility is that one of the intermediate states in yeast is related to the Grp94-like state described here, but this hypothesis will require further characterization of the yeast solution structure.
Our previous work27; 31
and the FRET studies demonstrate that nucleotide shifts the conformational equilibrium as opposed to explicitly determining the conformational state of Hsp90. An important difference between eukaryotes and prokaryotes may be the other factors that determine the conformational equilibrium. One hypothesis is that the equilibrium in eukaryotes has evolved to be controlled by client/cochaperone binding instead of pH. Bacteria may rely more heavily on the finely tuned conformational equilibrium to optimize the levels of both conformations because of the lack of cochaperones. It is also possible that the HtpG pH equilibrium acts as a sensor to tune chaperone response in times of metabolic stress. Identification of HtpG client proteins will assist in the development of in vivo
HtpG functional assays and provide information as to the types of client proteins that interact with each conformational state.
The functional differences of the different apo states observed here may also be relevant to other homologs. The question remains as to the generalizability of the preferential binding of the Grp94-like structure to client proteins. One possibility is that different substrates are recognized by different conformations of Hsp90, and this structural diversity is therefore one mechanism by which Hsp90 recognizes such a broad set of substrate proteins. All of these questions will require further studies of the yeast and human proteins and their interactions with client proteins. The results discussed here with HtpG provide critical insights into the mechanism of substrate activation and the differences in regulation between different species, and these results are an important step in better understanding the chaperone cycle of Hsp90.