Phospholamban and Sarcolipin Pentamers Naturally Associate with the Sarcoplasmic Reticulum Calcium Pump

Abstract

Phospholamban and sarcolipin interact with the sarcoplasmic reticulum calcium pump (SERCA) and regulate contractility in smooth, cardiac and skeletal muscle. While both proteins can form oligomers, it is thought that only the monomers interact with and inhibit SERCA. To address the role of the phospholamban and sarcolipin pentamers, we have studied their interaction with SERCA using electron cryo-microscopy of two-dimensional co-crystals. In our previous studies, phospholamban oligomers were found interspersed between SERCA dimers and we constructed a three-dimensional model of the complex. We also addressed the molecular characteristics of phospholamban that contribute to its interaction with SERCA and we examined the effects of phosphorylation and mutation of phospholamban on the structure of the complex with SERCA. In our recent work, we compared two crystal forms of SERCA in the absence and presence of phospholamban by electron cryo-microscopy - namely, small helical crystals and large two-dimensional crystals. The SERCA dimer ribbons that are found in both crystal forms consist of a rigid assembly of calcium-free SERCA molecules. While the lattice formed by the SERCA dimer ribbons is different in the helical and two-dimensional crystals, we show that a phospholamban oligomer interacts with SERCA in a similar manner in both crystal types. With this information, we next undertook a structural investigation of SERCA and sarcolipin in the two-dimensional crystals. A projection map was determined for SERCA in the presence of sarcolipin to a resolution of 8.5 A and was consistent with a pentameric state for sarcolipin. While both phospholamban and sarcolipin interacted with transmembrane segment M3 of SERCA, the interaction of the sarcolipin pentamer was mediated by an additional density consistent with a SLN monomer. We conclude that pentameric forms of both phospholamban and sarcolipin naturally associate with SERCA.