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

Sameer Varma ^*   and Eric Jakobsson ^*    ¶

^* Center for Biophysics and Computational Biology, National Center for Supercomputing Applications, Department of Biochemistry, Department of Molecular and Integrative Physiology, and ^¶ Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Correspondence: Address reprint requests to Eric Jakobsson, Tel.: 217-244-2896; Fax: 217-244-2909; E-mail: jake{at}ncsa.uiuc.edu.

Cytosolic phospholipase A₂ is involved in several signal transduction pathways where it catalyses release of arachidonic acid from intracellular 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 significantly 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 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 electron paramagnetic resonance data.