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Biochemical Characterization of the Interaction Between Hsp70s and Lipids

Seventy‐kilodalton heat shock proteins (Hsp70s) are molecular chaperones essential for maintaining cellular homeostasis and survival. Apart from their indispensable roles in protein homeostasis specific Hsp70s also localize at the plasma membrane and bind to specific lipids. The latter interaction has direct physiological outcomes including immune system activation, viral entry, lysosomal rescue, and promotion of apoptosis. Despite these essential functions the Hsp70‐lipid interactions remain largely uncharacterized. In this study, we characterized the interaction between HspA1A and lipids to understand the molecular mechanism of this interaction. We first established that although the nucleotide‐binding domain (NBD) of the protein is largely responsible for lipid‐binding and the substrate‐binding domain (SBD) does not directly bind to lipids, allosteric communication between the two domains is important for lipid‐binding. We then determined that a conformational change caused by nucleotide‐binding reduces lipid‐binding for a particular subset of lipids, while binding of protein substrates has no effect on the lipid‐binding. Next, we determined that the HspA1A‐lipid interaction is not purely electrostatic but it also depends on other forces. Furthermore, we found that HspA1A embeds in membranes when bound to specific lipids. We also established that protein embedding occurs via the SBD region of the protein. Based on these data we propose a model according to which the NBD domain of HspA1A contains two lipid binding sites, the first one targets and docks the protein to the membrane, while the second one facilitates the embedding of the SBD into the non‐polar interior of the membrane.