Atomic-Level Mechanisms for Phospholamban Regulation of the Calcium Pump |
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Authors: | L?Michel Espinoza-Fonseca Joseph?M Autry G?Lizbeth Ramírez-Salinas David?D Thomas |
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Institution: | 1.Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota;2.Laboratorio de Modelado Molecular y Bioinformática, Escuela Superior de Medicina, Instituto Politécnico Nacional, Mexico City, Mexico |
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Abstract: | We performed protein pKa calculations and molecular dynamics (MD) simulations of the calcium pump (sarcoplasmic reticulum Ca2+-ATPase (SERCA)) in complex with phospholamban (PLB). X-ray crystallography studies have suggested that PLB locks SERCA in a low-Ca2+-affinity E2 state that is incompatible with metal-ion binding, thereby blocking the conversion toward a high-Ca2+-affinity E1 state. Estimation of pKa values of the acidic residues in the transport sites indicates that at normal intracellular pH (7.1–7.2), PLB-bound SERCA populates an E1 state that is deprotonated at residues E309 and D800 yet protonated at residue E771. We performed three independent microsecond-long MD simulations to evaluate the structural dynamics of SERCA-PLB in a solution containing 100 mM K+ and 3 mM Mg2+. Principal component analysis showed that PLB-bound SERCA lies exclusively along the structural ensemble of the E1 state. We found that the transport sites of PLB-bound SERCA are completely exposed to the cytosol and that K+ ions bind transiently (≤5 ns) and nonspecifically (nine different positions) to the two transport sites, with a total occupancy time of K+ in the transport sites of 80%. We propose that PLB binding to SERCA populates a novel (to our knowledge) E1 intermediate, E1⋅H+771. This intermediate serves as a kinetic trap that controls headpiece dynamics and depresses the structural transitions necessary for Ca2+-dependent activation of SERCA. We conclude that PLB-mediated regulation of SERCA activity in the heart results from biochemical and structural transitions that occur primarily in the E1 state of the pump. |
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