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Sarcolipin and phospholamban inhibit the calcium pump by populating a similar metal ion-free intermediate state.

ABSTRACT: 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-?s 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-?s 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.

SUBMITTER: Espinoza-Fonseca LM

PROVIDER: S-EPMC4465059 | biostudies-literature | 2015 Jul 17-24

REPOSITORIES: biostudies-literature

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Sarcolipin and phospholamban inhibit the calcium pump by populating a similar metal ion-free intermediate state.

Espinoza-Fonseca L Michel LM Autry Joseph M JM Thomas David D DD

Biochemical and biophysical research communications 20150515 1-2

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 effi ...[more]

PMID: 25983321

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Project description:The sequential rise and fall of cytosolic calcium underlies the contraction-relaxation cycle of muscle cells. Whereas contraction is initiated by the release of calcium from the sarcoplasmic reticulum, muscle relaxation involves the active transport of calcium back into the sarcoplasmic reticulum. This reuptake of calcium is catalyzed by the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA), which plays a lead role in muscle contractility. The activity of SERCA is regulated by small membrane protein subunits, the most well-known being phospholamban (PLN) and sarcolipin (SLN). SLN physically interacts with SERCA and differentially regulates contractility in skeletal and atrial muscle. SLN has also been implicated in skeletal muscle thermogenesis. Despite these important roles, the structural mechanisms by which SLN modulates SERCA-dependent contractility and thermogenesis remain unclear. Here, we functionally characterized wild-type SLN and a pair of mutants, Asn4-Ala and Thr5-Ala, which yielded gain-of-function behavior comparable to what has been found for PLN. Next, we analyzed two-dimensional crystals of SERCA in the presence of wild-type SLN by electron cryomicroscopy. The fundamental units of the crystals are antiparallel dimer ribbons of SERCA, known for decades as an assembly of calcium-free SERCA molecules induced by the addition of decavanadate. A projection map of the SERCA-SLN complex was determined to a resolution of 8.5 Å, which allowed the direct visualization of an SLN pentamer. The SLN pentamer was found to interact with transmembrane segment M3 of SERCA, although the interaction appeared to be indirect and mediated by an additional density consistent with an SLN monomer. This SERCA-SLN complex correlated with the ability of SLN to decrease the maximal activity of SERCA, which is distinct from the ability of PLN to increase the maximal activity of SLN. Protein-protein docking and molecular dynamics simulations provided models for the SLN pentamer and the novel interaction between SERCA and an SLN monomer.

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Sarcolipin and phospholamban inhibit the calcium pump by populating a similar metal ion-free intermediate state.

Publications

Sarcolipin and phospholamban inhibit the calcium pump by populating a similar metal ion-free intermediate state.

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