Interaction of a sarcolipin pentamer and monomer with the sarcoplasmic reticulum calcium pump, SERCA

Abstract

The sequential rise and fall of cytosolic calcium underlies the contraction-relaxation cycle of muscle cells. While 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 re-uptake of calcium is catalysed by the sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA), which plays a lead role in muscle contractility. The activity of SERCA is regulated by small membrane protein subunits, 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, Asn 4 -Ala and Thr 5 -Ala, which yielded gainof-function behavior comparable to what has been found for PLN. Next, we analyzed twodimensional crystals of SERCA in the presence of wild-type SLN by electron cryo-microscopy.The fundamental units of the crystals are anti-parallel 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 a SLN pentamer. The SLN pentamer was found to interact with transmembrane segment M3 of SERCA, though the interaction appeared to be indirect and mediated by an additional density consistent with a 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 a SLN monomer. STATEMENT OF SIGNIFICANCEThis research article describes a novel complex of the sarcoplasmic reticulum calcium pump SERCA and its regulatory subunit sarcolipin. Given the potential role of sarcolipin in skeletal muscle non-shivering thermogenesis, the interactions between SERCA and sarcolipin are of critical importance. Using complementary approaches of functional analysis, electron crystallography, and molecular dynamics simulations, we demonstrate an inherent interaction 3 between SERCA, a sarcolipin monomer, and a sarcolipin pentamer. The interaction involves transmembrane segment M3 of SERCA, which allows sarcolipin to decrease the maximal activity or turnover rate of SERCA. Protein-protein docking and molecular dynamics simulations provided models for the SLN pentamer and the novel interaction between SERCA and a SLN monomer. 6 regulatory mechanism. However, the interactions of the PLN and SLN pentamers with SERCA are different and the functional effects on the maximal activity of SERCA are oppo...

Effects of amino acid modifications on the permeability of the pentameric sarcolipin channel

“…This study evaluated the structural dynamics of the pentameric complex, the SLN channel, with either a mutation or protonation of an amino acid. Pentameric channels are formed by self‐assembly and affect the ion permeability and anion selectivity under certain conditions 19,21 …”

“…Our previous works also suggested that the SLN monomers assembled into a stable oligomer by forming a repeated Leu‐Ile zipper in the TM region 20 . The channel‐like pentameric protein structure was visualized by cryo‐electron microscopy and FRET 21 . In the pentameric SLN channel structure, the pore has a hydrophilic‐inward surface that allows hydroxyl groups to interact positively with the infiltrated water molecules 22 .…”

Sarcolipin alters SERCA1a interdomain communication by impairing binding of both calcium and ATP

“…All these data on PLB could suggest that the same kind of interplay between SLN oligomerisation, palmitoylation and phosphorylation for inhibition of SERCA1a is present. Oligomerisation of SLN was recently proposed as a mechanism for SERCA1a regulation relying on the reduction of the interaction of both proteins similarly to PLB 55,56 . Even if the data presented in Fig.…”

“…PLB:Cys 36 is a residue predicted to be at the membrane interface as SLN:Cys9 3,15 . Sarcolipin also forms oligomers in detergent micelles, liposomes 57 and membranes 56 . Phosphorylation of SLN:Thr5 results in a loss of inhibition of SERCA1a 58 .…”

Protein Docking and Steered Molecular Dynamics Reveal Alternative Regulatory Sites on the SERCA Calcium Transporter

“…These data emphasize the role of M3 as a important site for PLB binding. Binding to M3 of SERCA has been observed for both PLB (29) and SLN (44) and is hypothesized to play a role in regulating the maximal activity (Vmax) of SERCA (45,46).…”