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. 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 Ca 2þ -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, Asn 4 -Ala and Thr 5 -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.

Sarcolipin Exhibits Abundant RNA Transcription and Minimal Protein Expression in Horse Gluteal Muscle

“…Oligomerization of synthetic horse SLN on SDS-PAGE suggests that self-association of horse SLN plays a role in the availability of SLN monomers for inhibition of horse SERCA. Binding of SLN and SERCA in a multimeric, pea-pod complex (SLN monomer, SLN pentamer, and SERCA monomer) is proposed to be an activating mechanism for the Ca 2+ activated ATPase function of SERCA [ 97 ].…”

Nothing Regular about the Regulins: Distinct Functional Properties of SERCA Transmembrane Peptide Regulatory Subunits

“…The N-terminus of SLN has a conserved threonine residue (Thr 5 ) that appears to be a target for phosphorylation by CaMKII [15] and serine/threonine kinase 16 (STK16 [16]). SLN is known to form a variety of oligomers with a monomer and dimer being the dominant species and the pentamer being less stable compared to PLN [35,57,58] (Figure 3A). SLN has also been shown to bind to the M3 accessory site of SERCA, though the interaction is distinct from PLN and involves both an SLN monomer and pentamer [58].…”

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 .…”