| The correct folding and assembly of many proteins depend on the help of molecular chaperones.Heat Shock Protein 90(Hsp90)is the most complex and important molecular chaperone in eukaryotes,which relies on ATP binding and hydrolysis to promote the maturation and activation of various substrates.More than 300 client proteins of Hsp90 have been identified,including various transcription factors,cell cycle regulators and,especially,kinases.Central questions to fully understanding the action of Hsp90 include how Hsp90 selectively recognize and interact with such a wide array of client proteins with no obvious consensus in sequences or structural motifs,and how Hsp90 utilized an ATP-fueled conformation change to fulfill its function.However,due to the inherent difficulties associates with the Hsp90 dynamics,these mechanisms remain poorly understood.Kinases represents one of the most important clients of the Hsp90,many of which have been closely related to cancer.In this study we report efforts to better understand the mechanism that how Hsp90 interacts with kinase client.To simplify the system,we chose the kinase HopBF1 as the client,a recently reported bacterial effector,and established a binary Hsp90-kinase interaction system in absence of any co-chaperones.We first confirmed the direct binding of Hsp90 to HopBF1 without the help of cochaperones by both ITC and NMR techniques,and further quantitatively characterized the interaction between the Hsp90 and HopBF1.Unexpectedly,our results show that the binding of HopBF 1 to Hsp90 is species specific,that is,HopBF1 only interacts with eukaryotic Hsp90(Hsp90β and Hsp82)and exhibits no binding to prokaryotic Hsp90(HTPG).To explore the molecular mechanism underlying,we built a series of truncation constructs of Hsp90β and,by a combination of ITC and NMR techniques,eventually identified the binding sites on Hsp90.Different from previous proposals,we show that HopBF1 binds to the hydrophobic regions at the interface between the N-terminal domain and the M-domain of Hsp90.Importantly,we found that the binding stoichiometry of Hsp90 to HopBF1 alters drastically at different nucleotide states of Hsp90,implying a client screening process in Hsp90 machinery.We further attempted to solve the complex structure of Hsp90-HopBF1 by employed cryo-electron microscopy and X-ray crystal diffraction techniques.However,despite of many trials,due to the intrinsic flexibility of this system,only low-resolution complex structure has been obtained.In conclusion,our study has established a binary Hsp90-kinase system and performed quantitative characterizations of the interaction between Hsp90 and a kinase for the first time.Our results demonstrate Hsp90 utilizes the interface region of its N-terminal and middle domain to interact with kinase client,a result distinct from previous studies.Moreover,we reveal that the binding stoichiometry of Hsp90 to HopBF1 changes dramatically during its chaperone cycle.Our results provide new insights into the chaperone mechanism of Hsp90. |
