The cPLA2 C2alpha domain in solution: structure and dynamics of its Ca2+-activated and cation-free states

Abstract

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 C2alpha 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 C2alpha 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 C2alpha domain in solution (equivalent to approximately 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.

FIGURE 1

Partial view of the structure of the C2 domain of cPLA₂ determined using (a) x-ray crystallography (12), and (b) NMR spectroscopy (16). The β-sheets are drawn as cartoons, the three CBLs are drawn as ribbons, and the coordinating side chains are highlighted as stick models. This spatial region of the domain binds Ca²⁺ ions and is also involved in membrane docking (2). We note two main differences between the structures obtained using x-ray crystallography and NMR spectroscopy: 1), the roughly helical segment of CBL-I in the NMR structure is a well-defined α-helix in the x-ray structure, and 2), the side chain of residue Asp-40 does not coordinate with either of the activating Ca²⁺ ions in the NMR structure as it does in the x-ray structure. This figure was made using PyMol (DeLano Scientific, Palo Alto, CA).

FIGURE 2

The structure of the helical segment of CBL-I as determined using (a) x-ray crystallography (12). The helical segment is drawn as a ribbon and the backbone atoms involved in intramolecular hydrogen bonds are highlighted as stick models. The hydrogen bonds between the backbone atoms of the ith and the (i + 4)th residues are drawn as dashed lines, and the distances between the proton donor and acceptor atoms are indicated in angstrom units. (b) NMR spectroscopy (16). The corresponding proton donors and acceptor atoms are shown and the distances between these atoms are also indicated in angstrom units. This figure was made using PyMol.

FIGURE 3

Coordinating side chains in the Ca²⁺-binding cleft of the domain. Snapshots were taken at every 0.5 ns of a 10-ns-long MD simulation of the cPLA₂ C2 domain and superimposed over each other. The side chains are drawn as stick models and the Ca²⁺ ions are drawn as spheres. We find that the crystallographic coordination scheme is maintained throughout the course of the simulation. This figure was made using PyMol.

FIGURE 4

RMS fluctuation of backbone atoms of the cPLA2 C2 domain. SPC, SPC/E, and TIP4P refer to RMS fluctuations of backbone atoms computed from 10-ns-long MD trajectories generated using those water models.

FIGURE 5

Time series of the distances between the four proton donor atoms (backbone nitrogens) and the four corresponding acceptor atoms (oxygens) belonging to the helical region of CBL-I. These distances were computed at every 10 ps of a 10-ns-long MD simulation of the cPLA₂ C2 domain in solution. (a) Wild-type domain. (b) G-36C mutant. (c) G-33T-G-36C mutant.

FIGURE 6

Ramachandran plots of the two glycine residues, G-33 and G-36, found in the helical segment of CBL-I of the cPLA2 C2 domain. These Ramachandran plots were computed from a 10-ns-long MD simulation of the domain in solution.

FIGURE 7

Time series of the RMS deviation of the backbone atoms of CBLs of the domain. The RMS deviation profiles of (a) CBL-I, (b) CBL-II, and (c) CBL-III, computed from the simulation of the cation-free state of the domain are compared to their corresponding RMS deviation profiles computed from the simulation of the Ca²⁺-bound state of the domain.

FIGURE 8

Evolution of the distances of the side-chain amino and carbonyl groups of residues Asn-65 and Asn-95, and the side-chain carboxylate groups of Asp-40 and Asp-93 from the side-chain carboxylate of Asp-43.

FIGURE 9

Ca²⁺-binding cleft of the cPLA₂ C2 domain in the absence of bound calcium. This structure was generated by replacing Ca²⁺ ions in the binding cleft with water molecules and then carrying out 1 ns of MD. We see that the secondary structure of the binding cleft is essentially unchanged. The large negative potential in the binding cleft created as a result of the removal of the two crystallographically resolved Ca²⁺ ions is released via three well-defined side-chain motions: χ-angle rotations (indicated) of the aspargines Asn-65 and -95 and a movement of Asp-40 away from the binding cleft.