Location of the Membrane-Docking Face on the Ca²⁺-Activated C2 Domain of Cytosolic Phospholipase A₂

Abstract

Docking of C2 domains to target membranes is initiated by the binding of multiple Ca2+ ions to a conserved array of residues imbedded within three otherwise variable Ca2+-binding loops. We have located the membrane-docking surface on the Ca2+-activated C2 domain of cPLA2 by engineering a single cysteine substitution at 16 different locations widely distributed across the domain surface, in each case generating a unique attachment site for a fluorescein probe. The environmental sensitivity of the fluorescein-labeled cysteines enabled identification of a localized region that is perturbed by Ca2+ binding and membrane docking. Ca2+ binding to the domain altered the emission intensity of six fluoresceins in the region containing the Ca2+-binding loops, indicating that Ca2+-triggered environmental changes are localized to this region. Similarly, membrane docking increased the protonation of six fluoresceins within the Ca2+-binding loop region, indicating that these three loops also are directly involved in membrane docking. Furthermore, iodide quenching measurements revealed that membrane docking sequesters three fluorescein labeling positions, Phe35, Asn64, and Tyr96, from collisions with aqueous iodide ion. These sequestered residues are located within the identified membrane-docking region, one in each of the three Ca2+-binding loops. Finally, cysteine substitution alone was sufficient to dramatically reduce membrane affinity only at positions Phe35 and Tyr96, highlighting the importance of these two loop residues in membrane docking. Together, the results indicate that the membrane-docking surface of the C2 domain is localized to the same surface that cooperatively binds a pair of Ca2+ ions, and that the three Ca2+-binding loops themselves provide most or all of the membrane contacts. These and other results further support a general model for the membrane specificity of the C2 domain in which the variable Ca2+-binding loops provide headgroup recognition at a protein-membrane interface stabilized by multiple Ca2+ ions.

C2 Domain of Protein Kinase Cα: Elucidation of the Membrane Docking Surface by Site-Directed Fluorescence and Spin Labeling

“…The nearly parallel membrane docking orientation observed for PKCα C2 domain is significantly different from the docking angle previously observed for the cPLA 2 C2 domain, which is tilted away from the parallel orientation (19,24,26). In both models, the three Ca 2+ binding loops dominate the membrane docking surface with the first and third Ca 2+ binding loops penetrating into the membrane.…”

The Molecular Basis of Differential Subcellular Localization of C2 Domains of Protein Kinase C-α and Group IVa Cytosolic Phospholipase A2

“…Mutational (17,19,23,24,26,28), labeling (49,50), and structural (12-14) studies of C2 domains have identified the residues in the Ca 2ϩ binding loops that play a key role in membrane binding. The present study shows that cationic residues, Arg 249 and Arg 252 , in Ca 2ϩ binding loops of PKC-␣-C2 are involved in its binding to the anionic membrane surface, whereas Asn 189 plays a critical role in PS selectivity.…”

Ca2+ Activation of the cPLA2 C2 Domain: Ordered Binding of Two Ca2+ Ions with Positive Cooperativity

“…Cys mutants were expressed as inclusion bodies in Escherichia coli, then were isolated, denatured, and refolded (26). Following refolding, each mutant was purified by FPLC size exclusion on a Superdex G-75 gel filtration column in purification buffer containing 100 mM KCl, 20 mM Tris pH 7.8 with HCl, and 1 mM CaCl 2 .…”