SELF-INDUCED DOCKING SITE OF A DEEPLY EMBEDDED PERIPHERAL MEMBRANE PROTEIN

¹ University of California, Irvine
² Univ. of Colorado
³ Univ. of California at Irvine

^* To whom correspondence should be addressed. E-mail: blanco{at}helium.biomol.uci.edu.

Submitted on June 6, 2006
Revised on July 17, 2006
Accepted on 5 October 2006

	Abstract

As a first step toward understanding the principles of the targeting of C2 domains to membranes, we have carried out a molecular dynamics (MD) simulation of the C2 domain of cytosolic phospholipase A2 (cPLA2-C2) in a 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayer at constant pressure and temperature (NpT: 300K and 1 atm). Using the high-resolution crystal structure of cPLA2-C2 as a starting point, we embedded two copies of the C2 domain into a pre-equilibrated membrane at the depth and orientation previously defined by electron paramagnetic resonance (EPR). Noting that in the membrane-bound state the three calcium binding loops (CBLs) are complexed to two calcium ions, we initially restrained the calcium ions at the membrane depth determined by EPR. But the depth and orientation of the domains remained within EPR experimental errors when the restraints were later removed. We find that the thermally disordered, chemically heterogeneous interfacial zones of phosphatidylcholine (PC) bilayers allow local lipid remodeling to produce a nearly perfect match to the shape and polarity of the C2 domain, thereby enabling the C2 domain to assemble and optimize its own lipid docking site. The result is a cup-like docking site with a hydrophobic bottom and hydrophilic rim. Contrary to expectations, we did not find direct interactions between the protein-bound calcium ions and lipid headgroups, which were sterically excluded from the calcium binding cleft. Rather, the lipid phosphate groups provided outer-sphere calcium coordination through intervening water molecules. These results show that the combined use of high resolution protein structures, EPR measurements, and MD simulations provides a general approach for analyzing the molecular interactions between membrane-docked proteins and lipid bilayers.

Key Words: C2 domain, EPR, cPLA2, molecular dynamics, signaling

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