Hsp90 couples ATP hydrolysis to large conformational changes essential for activation of client proteins. The structural transitions involve dimerization of the N-terminal domains and formation of 'closed states' involving the N-terminal and middle domains. Here, we used Hsp90 mutants that modulate ATPase activity and biological function as probes to address the importance of conformational cycling for Hsp90 activity. We found no correlation between the speed of ATP turnover and the in vivo activity of Hsp90: some mutants with almost normal ATPase activity were lethal, and some mutants with lower or undetectable ATPase activity were viable. Our analysis showed that it is crucial for Hsp90 to attain and spend time in certain conformational states: a certain dwell time in open states is required for optimal processing of client proteins, whereas a prolonged population of closed states has negative effects. Thus, the timing of conformational transitions is crucial for Hsp90 function and not cycle speed.
As an alternative pathway of controlled cell death, necroptosis can be triggered by tumor necrosis factor via the kinases RIPK1/RIPK3 and the effector protein mixed-lineage kinase domain-like protein (MLKL). Upon activation, MLKL oligomerizes and integrates into the plasma membrane via its executioner domain. Here, we present the X-ray and NMR costructures of the human MLKL executioner domain covalently bound via Cys86 to a xanthine class inhibitor. The structures reveal that the compound stabilizes the interaction between the auto-inhibitory brace helix α6 and the four-helix bundle by stacking to Phe148. An NMR-based functional assay observing the conformation of this helix showed that the F148A mutant is unresponsive to the compound, providing further evidence for the importance of this interaction. Real-time and diffusion NMR studies demonstrate that xanthine derivatives inhibit MLKL oligomerization. Finally, we show that the other well-known MLKL inhibitor Necrosulfonamide, which also covalently modifies Cys86, must employ a different mode of action.
The eukaryotic Hsp90 chaperone machinery comprises many co-chaperones and regulates the conformation of hundreds of cytosolic client proteins. Therefore, it is not surprising that the Hsp90 machinery has become an attractive therapeutic target for diseases such as cancer. The compounds used so far to target this machinery affect the entire Hsp90 system. However, it would be desirable to achieve a more selective targeting of Hsp90-co-chaperone complexes. To test this concept, in this-proof-of-principle study, we screened for modulators of the interaction between Hsp90 and its co-chaperone Aha1, which accelerates the ATPase activity of Hsp90. A FRET-based assay that monitored Aha1 binding to Hsp90 enabled identification of several chemical compounds modulating the effect of Aha1 on Hsp90 activity. We found that one of these inhibitors can abrogate the Aha1-induced ATPase stimulation of Hsp90 without significantly affecting Hsp90 ATPase activity in the absence of Aha1. NMR spectroscopy revealed that this inhibitory compound binds the N-terminal domain of Hsp90 close to its ATP-binding site and overlapping with a transient Aha1-interaction site. We also noted that this inhibitor does not dissociate the Aha1-Hsp90 complex but prevents the specific interaction with the N-terminal domain of Hsp90 required for catalysis. In consequence, the inhibitor affected the activation and processing of Hsp90-Aha1-dependent client proteins We conclude that it is possible to abrogate a specific co-chaperone function of Hsp90 without inhibiting the entire Hsp90 machinery. This concept may also hold true for other co-chaperones of Hsp90.
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