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Conformational Transitions and Alternating-Access Mechanism in the Sarcoplasmic Reticulum Calcium Pump.

ABSTRACT: Ion pumps are integral membrane proteins responsible for transporting ions against concentration gradients across biological membranes. Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), a member of the P-type ATPases family, transports two calcium ions per hydrolyzed ATP molecule via an "alternating-access" mechanism. High-resolution crystallographic structures provide invaluable insight on the structural mechanism of the ion pumping process. However, to understand the molecular details of how ATP hydrolysis is coupled to calcium transport, it is necessary to gain knowledge about the conformational transition pathways connecting the crystallographically resolved conformations. Large-scale transitions in SERCA occur at time-scales beyond the current reach of unbiased molecular dynamics simulations. Here, we overcome this challenge by employing the string method, which represents a transition pathway as a chainofstates linking two conformational endpoints. Using a multiscale methodology, we have determined all-atom transition pathways for three main conformational transitions responsible for the alternating-access mechanism. The present pathways provide a clear chronology and ordering of the key events underlying the active transport of calcium ions by SERCA. Important conclusions are that the conformational transition that leads to occlusion with bound ATP and calcium is highly concerted and cooperative, the phosphorylation of Asp351 causes areorganization of the cytoplasmic domains that subsequently drives the opening of the luminal gate, and thereclosing of luminal gate induces a shift in the cytoplasmic domains that subsequently enables the dephosphorylation of Asp351-P. Formation of transient residue-residue contacts along the conformational transitions predicted by the computations provide an experimental route to test the general validity of the computational pathways.

SUBMITTER: Das A

PROVIDER: S-EPMC5467534 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Conformational Transitions and Alternating-Access Mechanism in the Sarcoplasmic Reticulum Calcium Pump.

Das Avisek A Rui Huan H Nakamoto Robert R Roux Benoît B

Journal of molecular biology 20170116 5

Ion pumps are integral membrane proteins responsible for transporting ions against concentration gradients across biological membranes. Sarco/endoplasmic reticulum Ca<sup>2+</sup>-ATPase (SERCA), a member of the P-type ATPases family, transports two calcium ions per hydrolyzed ATP molecule via an "alternating-access" mechanism. High-resolution crystallographic structures provide invaluable insight on the structural mechanism of the ion pumping process. However, to understand the molecular detail ...[more]

PMID: 28093226

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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.

Dataset Information

Conformational Transitions and Alternating-Access Mechanism in the Sarcoplasmic Reticulum Calcium Pump.

Publications

Conformational Transitions and Alternating-Access Mechanism in the Sarcoplasmic Reticulum Calcium Pump.

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