Membrane-Docking Loops of the cPLA2 C2 Domain: Detailed Structural Analysis of the Protein-Membrane Interface via Site-Directed Spin-Labeling

Cellular signals are transmitted by a variety of mechanisms, including the release of small molecule second messengers such as Ca2+ or phosphoinositides, protein translocation between cellular compartments, and protein posttranslational modification. C2 domains are protein-signaling modules found in numerous signaling proteins (1–11). Usually, the function of such C2 domains is to trigger the translocation of proteins to specific cellular membranes in response to a Ca2+ signal. This targeting increases the likelihood of interaction of the signaling protein with its downstream target, often a membrane-bound lipid or protein. Cytosolic phospholipase A2 (cPLA2)1 hydrolyzes lipids containing arachadonic acid and thereby releases this important precursor from nuclear and endoplasmic reticulum (ER) membranes in response to a Ca2+ signal (12). The protein consists of two structurally distinct domains, an N-terminal C2 domain, which binds two Ca2+ ions and triggers docking to phospholipid membranes, and a C-terminal catalytic domain, which hydrolyzes the sn-2 ester of arachidonic acid-containing phospholipids (13). The protein initiates pathways that synthesize prostaglandins and trigger inflammation and that synthesize leukotrienes released as powerful chemoattractants (14, 15). Previous studies have characterized a number of Ca2+-regulated C2 domains that bind multiple Ca2+ ions and trigger docking to phospholipids on the surfaces of cellular membranes (3, 7, 16–20). Most of these domains employ an electrostatic mechanism of docking, and as a result, they require anionic lipids such as phosphatidylserine (PS) for membrane docking. In contrast, the C2 domain of cytosolic phospholipase A2 (cPLA2) employs a hydrophobic mechanism to dock to membrane surfaces containing neutral phospholipids such as phosphatidylcholine (PC). Since the cPLA2 C2 domain is the first characterized example of a C2 domain that exhibits a hydrophobic docking mechanism, it is known to exhibit novel features of Ca2+ activation and membrane docking. These features are likely to be relevant to newly reported C2 and C2-like domains also proposed to use a hydrophobic docking mechanism, such as that of 5-lipoxygenase (21). Three high-resolution structures of the C2 domain of cPLA2 have been solved (22–24). Two of these structures define the isolated C2 domain in crystals or solution, respectively, while the third reveals the crystal structure of the domain in the context of the full-length protein. These structures have confirmed that the cPLA2 C2 domain, like other structurally characterized C2 domains (25–30), exhibits the classic eight-strand antiparallel β-sandwich of the C2 motif, with three interstrand Ca2+-binding loops that bind two Ca2+ ions at saturating Ca2+ concentrations (Figure 1). Separate equilibrium dialysis and fluorescence binding studies carried out under physiological ionic conditions have revealed that the isolated cPLA2 C2 domain binds two Ca2+ ions, both when free in solution and when docked to membranes (19). To date, this C2 domain has failed to crystallize in the absence of Ca2+ or in the presence of a phospholipid headgroup analogue; thus, crystallographic studies have not yet provided information about the structure of the domain either in the absence of Ca2+ or in the presence of lipids. NMR studies have detected Ca2+-triggered chemical shift changes in the Ca2+-binding loops but could not clarify whether these changes reflect a conformational change, a change in the electrostatic environment, or a change in loop dynamics. Figure 1 Structure of the C2 Domain of cPLA2. (A) The crystal structure of the C2 domain of cPLA2 is shown (22), with eight β -strands depicted by ribbons and two Ca2+ ions shown as spheres. (B) Enlarged view of the three Ca2+-binding loops (CBLs). Figure ... Previous NMR, fluorescence, and EPR reports have indicated that the Ca2+-binding loops of the cPLA2 C2 domain provide most or all of the major lipid contacts when the domain docks to membranes. NMR studies have detected protein chemical shift changes localized primarily to the Ca2+-binding loops when the domain docks to the a lipid micelle (23). Fluorescence studies utilizing fluorescein probes coupled to engineered single-cysteine residues scattered over the protein surface have revealed direct fluorophore interactions between the Ca2+-binding loops and the membrane and have indicated that other regions of the protein surface do not contact the membrane (31). EPR studies using nitroxide spin probes coupled to cysteines scattered over the protein surface have also localized the membrane-binding surface to the Ca2+-binding loops (32, 58) and have defined an initial model for the depth of penetration of the domain into the membrane and its angular orientation relative to the membrane surface (32). These previous studies have raised important questions about the nature of the membrane-docking interaction. First, the information provided by scattered fluorescence- and spin-labeling sites could not rule out the possibility that the Ca2+-binding loops undergo major structural rearrangements upon Ca2+ binding and membrane docking. Moreover, the precision of the proposed EPR model for penetration depth and angular orientation was limited by the small number of positions examined in the Ca2+ and membrane-binding loops (only six out of 27 loop positions were examined (32)). The answers to these questions about the Ca2+-binding loops of the cPLA2 C2 domain could have broad implications since the Ca2+-binding loops of other C2 domains also dominate membrane-docking interactions (33–35). The present study introduces cysteines into all 27 positions of the three Ca2+-binding loops of the human cPLA2 C2 domain. This cysteine-scanning approach allows coupling of a thiol-specific spin-label to each cysteine for EPR spectroscopy (36). For 24 of the 27 positions, EPR spectra have been acquired for the free apo domain, the free Ca2+-occupied domain, and the Ca2+-occupied domain bound to unilamellar phospholipid vesicles composed of a physiological mixture of phosphatidylcholine (PC) and phosphati-dylserine (PS). The EPR spectra in the presence and absence of Ca2+ indicate that Ca2+ binding does not trigger major rearrangements of the Ca2+-binding loops. For the membrane-bound domains, continuous-wave power saturation measurements were used to calculate an EPR depth parameter for each spin-label in the loops. The resulting depth parameters indicate that the conformation of the Ca2+-binding loops docked to membranes is similar to their Ca2+-loaded conformation in the crystal and NMR structures. Finally, a novel and generalizable geometric analysis was applied to the extensive set of EPR depth parameters to more precisely determine the penetration depth and angular orientation of the domain with respect to the membrane surface. Notably, the interaction with the membrane is dramatically different than observed for C2 domains that exhibit an electrostatic docking mechanism.