A Functional, Pentameric Form of Phospholamban Is Required For Two-Dimensional Crystallization With the Sarcoplasmic Reticulum Calcium Pump

Abstract

A Functional, Pentameric Form Of Phospholamban Is Required For Two-Dimensional Crystallization With The Sarcoplasmic Reticulum Calcium PumpPhospholamban physically interacts with the sarcoplasmic reticulum calcium pump (also known as SERCA) and regulates 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 phospholamban. In our original work, phospholamban oligomers were found interspersed between dimer arrays of SERCA and a three-dimensional model was constructed to show potential interactions between the two proteins. In the present study, we have examined the effects of phospholamban phosphorylation and mutation on the formation of two-dimensional co-crystals with SERCA. Phospholamban phosphorylation at Ser16 and a well-characterized loss-of-function mutation (Asn34-to-Ala) significantly reduced crystal formation. More importantly, projection maps calculated from these crystals revealed that the densities attributable to phospholamban become disordered, suggesting a reduced interaction with SERCA. In contrast, a pentameric gain-of-function mutant (Lys27-to-Ala) significantly enhanced crystal formation. These latter crystals were used to calculate an improved projection map from frozen-hydrated crystals to a resolution of 8 A. We conclude that the oligomeric state of phospholamban in the crystals is a pentamer, and that phosphorylation and mutation of phospholamban alter physical interactions in the crystals in a manner that is consistent with a functional association with SERCA. Combined, the data suggest that the pentameric state of phospholamban is not simply an inactive storage form.