Structural and Functional Analysis of the Middle Segment of Hsp90: Implications for ATP Hydrolysis and Client Protein and Cochaperone Interactions

Abstract

Activation of client proteins by the Hsp90 molecular chaperone is dependent on binding and hydrolysis of ATP, which drives a molecular clamp via transient dimerization of the N-terminal domains. The crystal structure of the middle segment of yeast Hsp90 reveals considerable evolutionary divergence from the equivalent regions of other GHKL protein family members such as MutL and GyrB, including an additional domain of new fold. Using the known structure of the N-terminal nucleotide binding domain, a model for the Hsp90 dimer has been constructed. From this structure, residues implicated in the ATPase-coupled conformational cycle and in interactions with client proteins and the activating cochaperone Aha1 have been identified, and their roles functionally characterized in vitro and in vivo.

Conformational dynamics of the molecular chaperone Hsp90

“…Even though in the Aha1:Hsp90 complex parts of the catalytic loop are disordered, the orientation is much more consistent with the loop conformation found in the closed state (Figure 12). Interestingly, the R380A mutant, which has a decreased ATPase rate, is not activated by Aha1 (Meyer et al, 2003) implying that R380 is critical in the Aha1 mediated activation of Hsp90. The importance of the catalytic loop in Aha1 mediated activation is further highlighted by the insensitivity of the Hsp90 E381K (located on the catalytic loop) mutant to activation, even though the mutation alone has very little affect on the ATPase activity (Meyer et al, 2003).…”

“…Interestingly, the R380A mutant, which has a decreased ATPase rate, is not activated by Aha1 (Meyer et al, 2003) implying that R380 is critical in the Aha1 mediated activation of Hsp90. The importance of the catalytic loop in Aha1 mediated activation is further highlighted by the insensitivity of the Hsp90 E381K (located on the catalytic loop) mutant to activation, even though the mutation alone has very little affect on the ATPase activity (Meyer et al, 2003). All of the evidence suggests that Aha1 promotes or stabilizes a conformation of Hsp90 that occurs after NTD dimerization and has increased interactions between the MD and NTD.…”

“…For example, the binding of nucleotide leads to dimerization of the N-terminal domains and these changes represent a key step in the chaperone cycle of Hsp90. Other important conserved structural features include the lid (Figure 1, purple), a helix-loop-helix motif adjacent to the nucleotide binding pocket (Prodromou et al, 1997;Stebbins et al, 1997), and the catalytic loop (Figure 1, cyan), a region between the NTD and MD that is essential for catalysis (Meyer et al, 2003). These regions are also involved in the conformational dynamics of Hsp90, and the details of these structural changes and their importance in function are discussed in the following sections.…”

“…Cross-linked NTDs lacking the MD exhibit 100 fold lower ATPase activity than WT (Wegele et al, 2003), supporting a catalytic role for the MD. Residues in the MD near the interface with the NTD (the catalytic loop, Hsp82 residues 375-388) are essential for the hydrolysis of ATP (Meyer et al, 2003), and NTD dimerization serves to orient the catalytic loop so that hydrolysis can occur. Catalysis at both NTDs is not required for full activity as a heterodimer containing one WT NTD and one NTD that is unable to bind ATP is fully active ) supporting the hypothesis that NTD dimerization establishes the catalytically active NM conformation.…”

“…Disruption of this network through mutagenesis leads to a loss in ATPase activity. R380, has previously been shown to be essential for the ATPase activity of yHsp90 (Meyer et al, 2003). In the AMPPNP-bound structure of yHsp90, R380 is the only residue contacting the γ-phosphate, and the hydrophobic network may serve to stabilize this interaction.…”