Halpin Jackson C, Street Timothy O
Department of Biochemistry Brandeis University, Waltham, MA 02453, USA.
Department of Biochemistry Brandeis University, Waltham, MA 02453, USA.
J Mol Biol. 2017 Sep 15;429(19):2918-2930. doi: 10.1016/j.jmb.2017.08.005. Epub 2017 Aug 17.
The ATPase cycle of the Hsp90 molecular chaperone is essential for maintaining the stability of numerous client proteins. Extensive analysis has focused on ATP-driven conformational changes of Hsp90; however, little is known about how Hsp90 operates under physiological nucleotide conditions in which both ATP and ADP are present. By quantifying Hsp90 activity under mixed nucleotide conditions, we find dramatic differences in ADP sensitivity among Hsp90 homologs. ADP acts as a strong ATPase inhibitor of cytosol-specific Hsp90 homologs, whereas organellular Hsp90 homologs (Grp94 and TRAP1) are relatively insensitive to the presence of ADP. These results imply that an ATP/ADP heterodimer of cytosolic Hsp90 is the predominant active state under physiological nucleotide conditions. ADP inhibition of human and yeast cytosolic Hsp90 can be relieved by the cochaperone aha1. ADP inhibition of bacterial Hsp90 can be relieved by bacterial Hsp70 and an activating client protein. These results suggest that altering ADP inhibition may be a mechanism of Hsp90 regulation. To determine the molecular origin of ADP inhibition, we identify residues that preferentially stabilize either ATP or ADP. Mutations at these sites can both increase and decrease ADP inhibition. An accounting of ADP is critically important for designing and interpreting experiments with Hsp90. For example, contaminating ADP is a confounding factor in fluorescence resonance energy transfer experiments measuring arm closure rates of Hsp90. Our observations suggest that ADP at physiological levels is important to Hsp90 structure, activity, and regulation.
热休克蛋白90（Hsp90）分子伴侣的ATP酶循环对于维持众多客户蛋白的稳定性至关重要。广泛的分析集中在Hsp90由ATP驱动的构象变化上；然而，对于Hsp90在ATP和ADP均存在的生理核苷酸条件下如何运作却知之甚少。通过量化混合核苷酸条件下的Hsp90活性，我们发现Hsp90同源物之间在ADP敏感性上存在显著差异。ADP作为胞质溶胶特异性Hsp90同源物的强效ATP酶抑制剂，而细胞器Hsp90同源物（Grp94和TRAP1）对ADP的存在相对不敏感。这些结果表明，胞质Hsp90的ATP/ADP异二聚体是生理核苷酸条件下的主要活性状态。辅助伴侣蛋白aha1可解除ADP对人和酵母胞质Hsp90的抑制作用。细菌Hsp70和一种激活型客户蛋白可解除ADP对细菌Hsp90的抑制作用。这些结果表明，改变ADP抑制作用可能是Hsp90的一种调节机制。为了确定ADP抑制作用的分子起源，我们鉴定了优先稳定ATP或ADP的残基。这些位点的突变既能增加也能降低ADP抑制作用。对ADP的考量对于设计和解释Hsp90实验至关重要。例如，在测量Hsp90臂闭合速率的荧光共振能量转移实验中，污染的ADP是一个混杂因素。我们的观察结果表明，生理水平的ADP对Hsp90的结构、活性和调节很重要。
