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Atomic-level mechanisms for phospholamban regulation of the calcium pump.

ABSTRACT: We performed protein pKa calculations and molecular dynamics (MD) simulations of the calcium pump (sarcoplasmic reticulum Ca(2+)-ATPase (SERCA)) in complex with phospholamban (PLB). X-ray crystallography studies have suggested that PLB locks SERCA in a low-Ca(2+)-affinity E2 state that is incompatible with metal-ion binding, thereby blocking the conversion toward a high-Ca(2+)-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 Mg(2+). 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 Ca(2+)-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.

SUBMITTER: Espinoza-Fonseca LM

PROVIDER: S-EPMC4390807 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Atomic-level mechanisms for phospholamban regulation of the calcium pump.

Espinoza-Fonseca L Michel LM Autry Joseph M JM Ramírez-Salinas G Lizbeth GL Thomas David D DD

Biophysical journal 20150401 7

We performed protein pKa calculations and molecular dynamics (MD) simulations of the calcium pump (sarcoplasmic reticulum Ca(2+)-ATPase (SERCA)) in complex with phospholamban (PLB). X-ray crystallography studies have suggested that PLB locks SERCA in a low-Ca(2+)-affinity E2 state that is incompatible with metal-ion binding, thereby blocking the conversion toward a high-Ca(2+)-affinity E1 state. Estimation of pKa values of the acidic residues in the transport sites indicates that at normal intra ...[more]

PMID: 25863061

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Project description:We have performed microsecond molecular dynamics (MD) simulations and protein pKa calculations of the muscle calcium pump (sarcoplasmic reticulum Ca(2+)-ATPase, SERCA) in complex with sarcolipin (SLN) to determine the mechanism by which SLN inhibits SERCA. SLN and its close analog phospholamban (PLN) are membrane proteins that regulate SERCA by inhibiting Ca(2+) transport in skeletal and cardiac muscle. Although SLN and PLB binding to SERCA have different functional outcomes on the coupling efficiency of SERCA, both proteins decrease the apparent Ca(2+) affinity of the pump, suggesting that SLN and PLB inhibit SERCA by using a similar mechanism. Recently, MD simulations showed that PLB inhibits SERCA by populating a metal ion-free, partially-protonated E1 state of the pump, E1· [Formula: see text] . X-ray crystallography studies at 40-80 mM Mg(2+) have proposed that SLN-bound SERCA populates E1·Mg(2+), an intermediate with Mg(2+) bound near transport site I. To test this proposed mode of SLN regulation, we performed a 0.5-μs MD simulation of E1·Mg(2+)-SLN in a solution containing 100 mM K(+) and 3 mM Mg(2+), with calculation of domain dynamics in the cytosolic headpiece and side-chain ionization and occupancy in the transport sites. We found that SLN increases the distance between residues E771 and D800, thereby rendering E1·Mg(2+) incapable of producing a competent Ca(2+) transport site I. Following removal of Mg(2+,) a 2-μs MD simulation of Mg(2+)-free SERCA-SLN showed that Mg(2+) does not re-bind to the transport sites, indicating that SERCA-SLN does not populate E1·Mg(2+) at physiological conditions. Instead, protein pKa calculations indicate that SLN stabilizes a metal ion-free SERCA state (E1· [Formula: see text] ) protonated at residue E771, but ionized at E309 and D800. We conclude that both SLN and PLB inhibit SERCA by populating a similar metal ion-free intermediate state. We propose that (i) this partially-protonated intermediate serves as the consensus mechanism for SERCA inhibition by other members of the SERCA regulatory subunit family including myoregulin and sarcolamban, and (ii) this consensus mechanism is utilized to regulate Ca(2+) transport in skeletal and cardiac muscle, with important implications for therapeutic approaches to muscle dystrophy and heart failure.

Project description:We have performed molecular dynamics (MD) simulations to elucidate, in atomic detail, the mechanism by which the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) is activated by Ca(2+). Crystal structures suggest that activation of SERCA occurs when the cytoplasmic head-piece, in an open (E1) conformation stabilized by Ca(2+), undergoes a large-scale open-to-closed (E1 to E2) transition that is induced by ATP binding. However, spectroscopic measurements in solution suggest that these structural states (E1 and E2) are not tightly coupled to biochemical states (defined by bound ligands); the closed E2 state predominates even in the absence of ATP, in both the presence and absence of Ca(2+). How is this loose coupling consistent with the high efficiency of energy transduction in the Ca(2+)-ATPase? To provide insight into this question, we performed long (500 ns) all-atom MD simulations starting from the open crystal structure, including a lipid bilayer and water. In both the presence and absence of Ca(2+), we observed a large-scale open-to-closed conformational transition within 400 ns, supporting the weak coupling between structural and biochemical states. However, upon closer inspection, it is clear that Ca(2+) is necessary and sufficient for SERCA to reach the precise geometrical arrangement necessary for activation of ATP hydrolysis. Contrary to suggestions from crystal structures, but in agreement with solution spectroscopy, the presence of ATP is not required for this activating transition. Principal component analysis showed that Ca(2+) reshapes the free energy landscape of SERCA to create a path between the open conformation and the activated closed conformation. Thus the malleability of the free energy landscape is essential for SERCA efficiency, ensuring that ATP hydrolysis is tightly coupled to Ca(2+) transport. These results demonstrate the importance of real-time dynamics in the formation of catalytically competent conformations of SERCA, with broad implications for understanding enzymatic catalysis in atomic detail.

Dataset Information

Atomic-level mechanisms for phospholamban regulation of the calcium pump.

Publications

Atomic-level mechanisms for phospholamban regulation of the calcium pump.

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