Abstract
Hsp90 is a molecular chaperone that undergoes a dramatic ATP-dependent conformational change that is essential for its biological function. However, the structural details that link Hsp90 conformation to its essential ATPase activity are not yet understood. Here we discover that the bacterial Hsp90 (HtpG) has a pH-dependent ATPase activity that is unique among other Hsp90 homologs. The HtpG ATPase kcat and Km both increase with increasing pH. We identify a single histidine, H255, that is largely responsible for this striking behavior. Interestingly, we observe that H225 contributes to both the kcat and Km and that these changes are positively correlated over a wide range of pH values. H255 makes a direct contact to a negatively charged phosphate group on the bound nucleotide that can only be formed in the open, ATPase inactive, configuration of HtpG. These findings suggest a pH-dependent linkage between HtpG conformation and its ATPase activity. Indeed, we constructed a linkage model that captures the qualitative behavior of our experimentally measured pH-dependent kcat and Km values. This linkage model predicts a positive correlation between kcat and Km, as we observe in our experimental data. This is the first predictive model that connects Hsp90 structure to specific molecular contacts and their influence on the ATPase rate.