Microsecond molecular dynamics simulations of Mg²⁺- and K⁺-bound E1 intermediate states of the calcium pump.
We have performed microsecond molecular dynamics (MD) simulations to characterize the structural dynamics of cation-bound E1 intermediate states of the calcium pump (sarcoendoplasmic reticulum Ca²⁺-ATPase, SERCA) in atomic detail, including a lipid bilayer with aqueous solution on both sides. X-ray crystallography with 40 mM Mg²⁺ in the absence of Ca²⁺ has shown that SERCA adopts an E1 structure with transmembrane Ca²⁺-binding sites I and II exposed to the cytosol, stabilized by a single Mg²⁺ bound to a hybrid binding site I'. This Mg²⁺-bound E1 intermediate state, designated E1•Mg²⁺, is proposed to constitute a functional SERCA intermediate that catalyzes the transition from E2 to E1•2Ca²⁺ by facilitating H⁺/Ca²⁺ exchange. To test this hypothesis, we performed two independent MD simulations based on the E1•Mg²⁺ crystal structure, starting in the presence or absence of initially-bound Mg²⁺. Both simulations were performed for 1 µs in a solution containing 100 mM K⁺ and 5 mM Mg²⁺ in the absence of Ca²⁺, mimicking muscle cytosol during relaxation. In the presence of initially-bound Mg²⁺, SERCA site I' maintained Mg²⁺ binding during the entire MD trajectory, and the cytosolic headpiece maintained a semi-open structure. In the absence of initially-bound Mg²⁺, two K⁺ ions rapidly bound to sites I and I' and stayed loosely bound during most of the simulation, while the cytosolic headpiece shifted gradually to a more open structure. Thus MD simulations predict that both E1•Mg²⁺ and E•2K+ intermediate states of SERCA are populated in solution in the absence of Ca²⁺, with the more open 2K+-bound state being more abundant at physiological ion concentrations. We propose that the E1•2K⁺ state acts as a functional intermediate that facilitates the E2 to E1•2Ca²⁺ transition through two mechanisms: by pre-organizing transport sites for Ca²⁺ binding, and by partially opening the cytosolic headpiece prior to Ca²⁺ activation of nucleotide binding.
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America.
L Michel
LM
Espinoza-Fonseca
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America.
Joseph M
JM
Autry
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota, United States of America.
David D
DD
Thomas
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D018360:Crystallography, X-Ray; D003600:Cytosol / Q000201:enzymology; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D011188:Potassium / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors
D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D008274:Magnesium; D011188:Potassium; D002118:Calcium