The molecular basis of ceramide-1-phosphate recognition by C2 domains

Abstract

Group IVA cytosolic phospholipase A 2 (cPLA 2), which harbors an N-terminal lipid binding C2 domain and a C-terminal lipase domain, produces arachidonic acid from the sn-2 position of zwitterionic lipids such as phosphatidylcholine. The C2 domain has been shown to bind zwitterionic lipids, but more recently, the anionic phosphomonoester sphingolipid metabolite ceramide-1-phosphate (C1P) has emerged as a potent bioactive lipid with high affi nity for a cationic patch in the C2 domain-groove. To systematically analyze the role that C1P plays in promoting the binding of cPLA 2-C2 to biological membranes, we employed biophysical measurements and cellular translocation studies along with mutagenesis. Biophysical and cellular translocation studies demonstrate that C1P specifi city is mediated by Arg 59 , Arg 61 , and His 62 (an RxRH sequence) in the C2 domain. Computational studies using molecular dynamics simulations confi rm the origin of C1P specifi city, which results in a spatial shift of the C2 domain upon membrane docking to coordinate the small C1P headgroup. Additionally, the hydroxyl group on the sphingosine backbone plays an important role in the interaction with the C2 domain, further demonstrating the selectivity of the C2 domain for C1P over phosphatidic acid. Taken together, this is the fi rst study demonstrating the molecular origin of C1P recognition.Copyright © 2013 by the American Society for Biochemistry and Molecular Biology, Inc..