Effect of PIP2 Binding on the Membrane Docking Geometry of PKCα C2 Domain: An EPR Site-Directed Spin-Labeling and Relaxation Study†

Many diverse cell signaling processes rapidly modulate the levels of intracellular small molecule second messengers in order to spatially and temporally regulate the activity of downstream effectors. Important reactions on membrane surfaces are often stimulated by second messenger signals which recruit signaling enzymes to the appropriate membrane, thereby bringing these enzymes to their membrane-bound substrates. Such membrane recruitment is typically controlled by a membrane targeting domain activated by the binding of a second messenger, often a signaling lipid or cytoplasmic Ca2+. One the most prevalent membrane targeting motifs is the C2 domain, which can be activated by Ca2+ binding and is widely found in membrane-targeted signaling proteins [reviewed in refs 1–8]. In a typical cell, transient cytoplasmic Ca2+ signals recruit multiple C2 domain-containing proteins to specific intracellular membrane surfaces, thereby modulating crucial membrane-associated signaling pathways. The conventional isoforms of protein kinase C (PKCα,1 PKCβ and PKCγ) possess Ca2+-activated C2 domains which recruit their parent proteins specifically to the inner leaflet of the plasma membrane where they phosphorylate membrane-bound substrate proteins [reviewed in refs 7, 9–13]. Their shared topology consists of an N-terminal pseudosubstrate motif that provides kinase autoinhibition, followed by a pair of C1 domains that bind the signaling lipid diacylgycerol (DAG), then by a single plasma membrane-targeting C2 domain, and finally by the C-terminal kinase domain. The present study focuses on the C2 domain of the conventional PKCα protein, which binds two Ca2+ ions and associates with two lipids essential for its plasma membrane targeting: phosphatidylserine (PS), the most abundant anionic lipid of the plasma membrane; and phosphatidylinositol-4,5-bisphosphate (PIP2), the most abundant phosphorylated PIP lipid (14–24). The activation of conventional PKCs has been described as a sequential process in which, following a transient Ca2+ signal, the Ca2+-occupied C2 domain first associates with plasma membrane PS and PIP2, thereby allowing the C1 domains to search for the more rare DAG messenger (11, 24, 25). Ultimately, the simultaneous binding of the C1 and C2 domains to their lipid targets displaces the pseudosubstrate from the kinase active site. The ensuing loss of autoinhibition, together with close proximity to membrane-bound substrates, provides dual activation of the kinase domain. High resolution X-ray crystal structures are available for PKCα C2 domain and several of its complexes (16, 26). As for other C2 domains, the core of this domain is an eight-stranded antiparallel β-sandwich. At one edge of the sandwich lie three interstrand Ca2+- and membrane-binding loops (CMBL1–3). The crystal structure of a complex between PKCα C2 domain and the PS headgroup reveals that the two bound Ca2+ ions chelated by CMBL1–3 also receive direct and indirect coordination from the 1-phosphate of the PS headgroup (16), indicating that the Ca2+ binding site is central to PS recognition and binding. In addition, the structure of a different PS complex shows that a basic cluster of four lysine residues (K197, K199, K209, K211), all located on the β3-β4 hairpin, directly contacts a second PS head-group, indicating that this basic cluster serves as a second binding site for anionic lipics such as PS (26). Recently, it has been established that the lysine cluster binds PIP2 with higher affinity than PS, and that PIP2 binding is essential for high affinity membrane docking in vitro as well as specific plasma membrane targeting in live cells (20, 22–24). Despite advances in the structural analysis of isolated PKC C2 domains, the structures of their membrane-docked states remain poorly described. Of particular interest for the conventional PKC C2 domains is a structural understanding of their simultaneous docking to its two target lipids, PS and PIP2, on a bilayer surface. Currently, high resolution methods are not yet capable of analyzing the structures of peripheral proteins docked to lipid bilayers, thus medium resolution approaches must be employed to generate molecular pictures of peripheral proteins in their active, membrane-bound states. In recent years, an EPR approach involving site-directed spin labeling and spin relaxation measurements (27–29) has been shown to be an effective method for elucidating membrane docking geometries, and has been successfully applied to several C2 domains (30–35). The docking geometry provided by EPR analysis, in turn, can serve as an experimentally defined starting point for subsequent molecular dynamics simulations designed to develop atomic resolution models of the membrane-docked protein (36). Previously, in an initial study of the PKCα C2 domain, we used EPR and fluorescence spectroscopies to identify the membrane docking surfaces, and to develop a preliminary model for the membrane docking geometry (37). The resulting model of this C2 domain docked to 3:1 PC:PS membranes suggested that the CMBLs penetrate the headgroup region of the bilayer, while a cluster of lysine residues lies near the surface of the headgroup region. However, the simplified lipid mixture used in the model target membranes lacked the important target lipid PIP2 as well as other lipids found in the inner leaflet of the plasma membrane. Moreover, this previous study employed a low density of spin label positions on the membrane docking surface, yielding only a preliminary, qualitative picture of the membrane docking geometry. The present study utilizes EPR site-directed spin labeling and relaxation techniques to generate the first medium resolution structural model of the PKCα C2 domain docked to a lipid bilayer composed of a physiological mixture of lipids. Specifically, the membrane penetration depth and docking angle are determined for the C2 domain bound to physiological membranes lacking or containing the target lipid PIP2. The results provide the first molecular view of a peripheral membrane protein simultaneously docked to two target lipids, thereby significantly extending our mechanistic understanding of the dual lipid specificity required for plasma membrane targeting by conventional PKC C2 domains. Moreover, the findings reveal that PIP2 binding to the lysine cluster significantly alters the membrane docking geometry of the C2 domain by tilting the domain relative to the membrane surface. This advance demonstrates that the EPR approach can be used to analyze changes in membrane docking geometry triggered by protein switching between different functional states. Finally, the results imply that target lipid-triggered geometry changes could play an important role in modulating the bound state lifetime and function of membrane-bound, conventional PKCs.