Ca<sup>2+</sup> Stoichiometry Controls the Binding Mode of the PKCα C2 Domain to Anionic Membranes.

Abstract

The activation of the cell signaling enzyme protein kinase Cα (PKCα) requires the association of its N-terminal regulatory region to cell membranes containing signaling lipids such as diacylglycerol, phosphatidylserine (PS), and phosphatidylinositol 4,5-bisphosphate (PIP2). The C2 domain, one of the N-terminal regulatory domains, targets and binds to PS/PIP2-containing membranes in a Ca²⁺-dependent manner via its Ca²⁺-binding loops and lysine-rich cluster. Here, we utilized multiple replicas of highly mobile membrane mimetic (HMMM) simulations to investigate how the Ca²⁺-binding stoichiometry of PKCα controls membrane binding of the C2 domain. Our HMMM simulations revealed two distinct C2 membrane-binding modes with specific lipid interactions in response to different Ca²⁺-binding stoichiometries at the Ca²⁺-binding loops of the C2 domain. Electrostatic interactions between anionic lipids and Ca²⁺-binding loops/lysine-rich cluster account for driving the initial targeting of the C2 domain to membranes with PS and PIP2. Once the C2 domain is bound to the membranes, the Ca²⁺-binding stoichiometry at the Ca²⁺-binding loops alters the population of the two membrane-binding modes. Our results suggest that Ca²⁺-dependent signaling of PKCα activation might occur through modulation of its membrane-binding modes, which could in turn affect the overall modular organization of PKCα.