Que Nanette L S, Seidler Paul M, Aw Wen J, Chiosis Gabriela, Gewirth Daniel T
Hauptman Woodward Medical Research Institute, Buffalo, New York, USA.
Chemical Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
Proteins. 2025 Mar;93(3):654-672. doi: 10.1002/prot.26756. Epub 2024 Oct 29.
Grp94 is the endoplasmic reticulum paralog of the hsp90 family of chaperones, which have been targeted for therapeutic intervention via their highly conserved ATP binding sites. The design of paralog-selective inhibitors relies on understanding the protein structural elements that drive higher affinity in selective inhibitors. Here, we determined the structures of Grp94 and Hsp90 in complex with the Grp94-selective inhibitor PU-H36, and of Grp94 with the non-selective inhibitor PU-H71. In Grp94, PU-H36 derives its higher affinity by utilizing Site 2, a Grp94-specific side pocket adjoining the ATP binding cavity, but in Hsp90 PU-H36 occupies Site 1, a side pocket that is accessible in all paralogs with which it makes lower affinity interactions. The structure of Grp94 in complex with PU-H71 shows only Site 1 binding. While changes in the conformation of helices 4 and 5 in the N-terminal domain occur when ligands bind to Site 1 of both Hsp90 and Grp94, large conformational shifts that also involve helix 1 are associated with the engagement of the Site 2 pocket in Grp94 only. Site 2 in Hsp90 is blocked and its helix 1 conformation is insensitive to ligand binding. To understand the role of helix 1 in ligand selectivity, we tested the binding of PU-H36 and other Grp94-selective ligands to chimeric Grp94/Hsp90 constructs. These studies show that helix 1 is the major determinant of selectivity for Site 2 targeted ligands and also influences the rate of ATPase activity in Hsp90 paralogs.
Grp94是伴侣蛋白hsp90家族的内质网旁系同源物，其高度保守的ATP结合位点已成为治疗干预的靶点。旁系同源物选择性抑制剂的设计依赖于对驱动选择性抑制剂更高亲和力的蛋白质结构元件的理解。在这里，我们确定了与Grp94选择性抑制剂PU-H36结合的Grp94和Hsp90的结构，以及与非选择性抑制剂PU-H71结合的Grp94的结构。在Grp94中，PU-H36通过利用位点2获得更高的亲和力，位点2是Grp94特有的侧袋，毗邻ATP结合腔，但在Hsp90中，PU-H36占据位点1，这是一个在所有旁系同源物中都可接近的侧袋，与之形成较低亲和力的相互作用。与PU-H71结合的Grp94的结构仅显示位点1结合。虽然当配体与Hsp90和Grp94的位点1结合时，N端结构域中螺旋4和5的构象会发生变化，但仅在Grp94中，涉及螺旋1的大的构象变化与位点2口袋的结合有关。Hsp90中的位点2被阻断，其螺旋1构象对配体结合不敏感。为了理解螺旋1在配体选择性中的作用，我们测试了PU-H36和其他Grp94选择性配体与嵌合Grp94/Hsp90构建体的结合。这些研究表明，螺旋1是位点2靶向配体选择性的主要决定因素，也影响Hsp90旁系同源物中ATPase活性的速率。
