The cPLA₂ C2 Domain in Solution: Structure and Dynamics of its Ca²⁺-activated and Cation-free States

¹ Sandia National Laboratories
² University of Illinois

^* To whom correspondence should be addressed. E-mail: jake{at}ncsa.uiuc.edu.

Submitted on June 20, 2006
Revised on August 1, 2006
Accepted on 13 October 2006

	Abstract

Cytosolic phospholipase A₂ (cPLA₂) is involved in several signal transduction pathways where it catalyses release of arachidonic acid from intra-cellular lipid membranes. Its membrane insertion is facilitated by its independently folding C2 domain, which is activated by the binding of two intracellular Ca²⁺ ions. However, the details of its membrane insertion mechanism, including its Ca²⁺-activation mechanism, are not understood. There are several unresolved issues, including the following. There are two experimentally resolved structures of the Ca²⁺-activated state of its isolated C2 domain, one determined using x-ray crystallography and the other determined using NMR spectroscopy, which differ from each other substantially in the spatial region that inserts into the membrane. This by itself adds to ambiguities associated with investigations targeting its mechanism of membrane insertion. Furthermore, there is no experimentally determined structure of its cation free state, which hinders investigations associated with its cation activation mechanism. In this work, we generate several unrestrained molecular dynamics (MD) trajectories of its isolated C2 domain in solution (equivalent to ~60 ns), and investigate these issues. Our main results are as follows: (a) the Ca²⁺ coordination scheme of the domain is consistent with the x-ray structure and with previous mutagenesis studies, (b) the helical segment of the Ca²⁺ binding loop, CBL-I, undergoes nanosecond timescale flexing (but not an unwinding), as can be inferred from physiological temperature NMR data and in contrast to low temperature x-ray data, and (c) removal of the two activating Ca²⁺ ions from their binding pockets does not alter the backbone structure of the domain, a result consistent with EPR data.

Key Words: Ca2+ binding protein, Ca2+ signaling, electrostatics, ligand binding, protein disorder, protein folding