The Oligomeric Forms Of Phospholamban And Sarcolipin Physically Interact With The Sarcoplasmic Reticulum Calcium Pump

Abstract

Phospholamban and sarcolipin physically interact with the sarcoplasmic reticulum calcium pump (also known as SERCA) and regulate contractility of the heart in response to adrenergic stimuli. We have studied this interaction using electron microscopy of large two-dimensional crystals of SERCA in complex with either phospholamban or sarcolipin. The crystals are comprised of the anti-parallel dimer ribbons of SERCA molecules previously seen in helical crystals, but packed into a novel lattice with p22121 symmetry. In previous studies, phospholamban pentamers were found interspersed between the SERCA dimer ribbons and a three-dimensional model was constructed to show potential interactions with SERCA. Herein, we have obtained two-dimensional co-crystals of SERCA and sarcolipin. Our analyses indicate that the oligomeric states of phospholamban and sarcolipin are similar in the context of the crystals and are most consistent with a pentameric arrangement. We also examined the crystallization behavior of gain-of-function mutants of phospholamban (Lys27 to Ala) and sarcolipin (Asn4 to Ala) in an attempt to understand the physiological relevance of the crystal contacts. In both cases, the gain-of-function mutants enhance crystal formation, supporting the notion that the crystal contacts represent a functional interaction. This interaction occurs within the membrane and most likely involves transmembrane segment M3 of SERCA. Importantly, this transmembrane segment of SERCA bears homology with the Leu-Ile zipper found in phospholamban. The combined results suggest that SERCA reversibly dissociates the phospholamban and sarcolipin oligomers, actively influencing the pool of monomers available for the inhibitory interaction.