The regulation of the Hsp70 family molecular chaperone BiP via phosphorylation

As the sole member of the Hsp70 family found within the endoplasmic reticulum, BiP plays a critical role in the protein homeostasis of the ER in order to prevent build-up of unfolded proteins which would lead to ER stress. Due to this critical function and a complex allosteric cycle associated with it, the chaperone requires a complex multi-layered system of regulation. It is one aspect of this regulation, the limitedly characterised phosphorylation of BiP, which this study aimed to generate a greater understand of the mechanism of. In this regard, this study was able to identify and select two substrate binding domain located phosphorylation sites which were demonstrated to be only conserved within ER located Hsp70s (and non-conserved in Hsp70 located in other sub-cellular locations) based on conservational analysis of ER and cytoplasmic located Hsp70s. These sites were mutated to phosphomimetic mutations in order to simulate the effects of phosphorylation and characterise them. The study used a combination of methyl NMR to monitor changes in the conformational ensemble, biochemical assays to measure alterations to the rate of ATP hydrolysis and effects to the interactions with a known substrates IRE1 in the characterisation process. This approach allowed the characterisation of a regulation mechanism of BiP, in which phosphomimetic mutants and potentially phosphorylation caused a disruption of the domain undocked conformation whilst stabilising both the domain docked and intermediate transient conformation which results in the fine-tuning of BiP functions. These functions include increased ATPase activity as well as a reduced ability to deoligomerize IRE1. Additionally, the study was successful in the identification of the BiP binding site of IRE1-LD.