Exploring the interaction between HSPA5 and HSP70-escort protein 1 (HEP1): mechanistic, structural and functional insights

HSPA5 (BiP/Grp78) is a molecular chaperone member of the Heat Shock Protein 70 kDa (HSP70) family. The HSPA5 is constituted by the Substrate Binding Domain (SBD) connected to the Nucleotide Binding Domain (NBD), which together regulate the different structural conformations of the chaperone, as well as its functional cycle, due to their interaction with ATP and ADP nucleotides and their co-chaperone partners. Because it plays an essential role in protein homeostasis, HSPA5 has been studied as well in different pathologies, including cancer, neurodegenerative diseases and, more recently, Covid-19. Although it is mostly described as a resident of the endoplasmic reticulum, the presence of HSPA5 in cell subcompartments such as mitochondria, nucleoplasm and cytoplasm are reported. Here, combining cellular assays and biochemical methods, we report for the first time the ability of HSPA5 to interact with a mitochondrial co-chaperone known as HEP1 (HSP70 escort protein 1), thus demystifying the idea that HEP1 is an exclusive co-chaperone of mitochondrial HSP70 (HSPA9/mortalin). Our in vitro data suggest a HEP1-HSPA5 interaction thermodynamically favored by entropy and enthalpy, and our in silico data demonstrate that the interaction site directly involves either the HSPA5 NBD (HSPA5-N) and the hydrophobic core of the co-chaperone. Surprisingly, HEP1 did not alter the HSPA5 ATPase activity, however it prevented the propensity to auto-aggregation of HSPA5-N and retarding such a process. Finally, using biotin-dependent proximity identification (BioID) by cellular approaches, we now find that HEP1 is also capable of interacting with other proteins moreover to HSP70s and studies are underway to explore the functional implications of these interactions.