Influence of Dimeric Interactions on Voltage Sensing Phosphatase Activity

Abstract

Multimeric interactions in proteins are a very common and extremely important phenomenon. Signaling cascades depend on delicate balances of homo- or heteromeric protein interactions to execute cellular responses to stimuli. We recently showed that such homomeric interactions exist in the voltage sensing phosphatase (VSP) from Ciona intestinalis. VSP bridges two fundamental cell signals, voltage and phosphatidylinositol phosphate (PIPs) concentrations, through voltage regulated enzyme catalysis. The voltage sensing domain (VSD) from VSP senses membrane voltage changes triggering the cytosolic phosphatase domain (PD) to dephosphorylate PIPs. VSP activity directly links voltage to PIP concentrations, both of which are key components in cell excitability, migration and immune response. Hence, understanding the biophysics of VSP will play a critical role in identifying the biological context. Our recent study showed that VSP dimerizes predominantly through the VSD and that VSP dimers can act in a dominant-negative fashion (Rayaprolu et al, JGP, 2018). To further probe those dimeric interactions, we identified potential interacting residues from the crystal structures of both the VSD and PD of Ci-VSP. We made mutations targeting those residues and tested them for their ability to co-immunoprecipitate. Some appear to break the dimer while others may enhance the interactions. Next, we will confirm the presence or absence of a dimeric unit with these mutants using single molecule pulldown (SiMPull) experiments and subsequently characterize the voltage dependence and enzymatic activity using electrophysiology. These results may shed light on the function of the two human VSP homologs since one of them is endogenously inactive and suggests a physiologically relevant dominant-negative behavior. The functional impact of dimerization could ultimately reveal how VSPs may be involved in voltage dependent PIP signaling in cells.