TY - JOUR
T1 - The cPLA2 C2α domain in solution
T2 - Structure and dynamics of its Ca2+-activated and cation-free states
AU - Varma, Sameer
AU - Jakobsson, Eric
N1 - Funding Information:
This work was supported by National Institutes of Health grant GM R01-054651 to H.L.S./E.J., by a Dept. of Energy/Genomes to Life grant to E.J., and by National Institutes of Health grant 2PN2EY016570-02.
PY - 2007/2
Y1 - 2007/2
N2 - 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 C2α 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 Ca 2+-activated state of its isolated C2α 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 C2α 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 Ca 2+-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.
AB - 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 C2α 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 Ca 2+-activated state of its isolated C2α 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 C2α 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 Ca 2+-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.
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U2 - 10.1529/biophysj.106.091850
DO - 10.1529/biophysj.106.091850
M3 - Article
C2 - 17085504
AN - SCOPUS:33846795976
VL - 92
SP - 966
EP - 976
JO - Biophysical Journal
JF - Biophysical Journal
SN - 0006-3495
IS - 3
ER -