Interaction of HspBP1 with Hsp70

Abstract

The Hsp70 family protein members are involved in many diverse processes that are essential for cell survival. The Hsp70 cochaperones control and specify Hsp70 interactions by modifying Hsp70 activity and linking Hsp70 proteins to specific cellular processes. Understanding the mechanistic details of how cochaperones co-opt Hsp70 for specific functions in a cell may provide insight into the workings of specific cellular functions as well as a broader understanding of how multifunctional proteins are regulated. HspBP1 is an Hsp70 cochaperone that binds to the ATPase domain of Hsp70 and inhibits its ATPase activity. The purpose of these studies was to gain a basic understanding of the Hsp70/HspBP1 interaction. The first goal was to define the structural domains of HspBP1 and explore the secondary structure of those domains. The next goal was to determine if HspBP1's ability to bind to Hsp70 and inhibit its activity segregated to its distinct protein domains. Finally, the effects of HspBP1 association on Hsp70 structure were investigated. It was determined from these studies that although HspBP1 is encoded by seven exons, it has only two structural domains as determined by limited proteolysis. Domain I, amino acids 1-83, is largely unstructured. Domain II, amino acids 84-359 is predicted to be 43% helical using circular dichroism. It was also shown that domain II is sufficient for binding to the Hsp70 ATPase domain and inhibiting luciferase renaturation although domain I was required for full activity. These studies also describe a novel activity for HspBP1, the ability to change the conformation of the ATPase domain. Only domain II of HspBP1 is required to bring about this conformational change. The studies presented here are the first to examine HspBP1's structure and determine how its structural domains interact with Hsp70. Prior to these studies, no structural information had been reported for an HspBPl family member. This information, along with the discovery that HspBPl can alter the conformation of the ATPase domain of Hsp70 will bring us closer to understanding this protein family's role in Hsp70 regulation.