Hsp72 is a member of the 70-kDa heat shock family of molecular chaperones (Hsp70s) that comprise a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD) connected by a linker that couples the exchange of adenosine diphosphate (ADP) for adenosine triphosphate (ATP) with the release of the protein substrate. Mitotic phosphorylation of Hsp72 by the kinase NEK6 at Thr⁶⁶ located in the NBD promotes the localization of Hsp72 to the mitotic spindle and is required for efficient spindle assembly and chromosome congression and segregation. We determined the crystal structure of the Hsp72 NBD containing a genetically encoded phosphoserine at position 66. This revealed structural changes that stabilized interactions between subdomains within the NBD. ATP binding to the NBD of unmodified Hsp72 resulted in the release of substrate from the SBD, but phosphorylated Hsp72 retained substrate in the presence of ATP. Mutations that prevented phosphorylation-dependent subdomain interactions restored the connection between ATP binding and substrate release. Thus, phosphorylation of Thr⁶⁶ is a reversible mechanism that decouples the allosteric connection between nucleotide binding and substrate release, providing further insight into the regulation of the Hsp70 family. We propose that phosphorylation of Hsp72 on Thr⁶⁶ by NEK6 during mitosis promotes its localization to the spindle by stabilizing its interactions with components of the mitotic spindle.

Hsp72是分子伴侣蛋白（Hsp70）的70 kDa热休克家族的成员，该分子伴侣包含一个核苷酸结合结构域（NBD）和一个底物结合结构域（SBD），该结构域通过一个连接二磷酸腺苷（ADP）交换的接头连接）释放三磷酸腺苷（ATP），释放出蛋白质底物。Thr ^66时激酶NEK6使Hsp72的有丝分裂磷酸化位于NBD内的Hsp72促进了Hsp72定位于有丝分裂纺锤体，是高效纺锤体组装以及染色体聚集和分离所必需的。我们确定了Hsp72 NBD的晶体结构，该基因在位置66处含有遗传编码的磷酸丝氨酸。这揭示了稳定NBD子域之间相互作用的结构变化。ATP与未修饰的Hsp72的NBD结合导致底物从SBD释放，但是磷酸化的Hsp72在ATP存在下保留了底物。阻止磷酸化依赖性亚域相互作用的突变恢复了ATP结合与底物释放之间的联系。因此，Thr ^66的磷酸化是一种可逆的机制，可将核苷酸结合与底物释放之间的变构关系解耦，从而进一步了解Hsp70家族的调控。我们提出有丝分裂期间NEK6在Thr ⁶⁶上对Hsp72的磷酸化通过稳定其与有丝分裂纺锤体成分的相互作用而促进了其对纺锤体的定位。