Structural and Functional Studies of Phospholamban-Sarcolipin Chimeras

Abstract

The sarcoplasmic reticulum (SR) is a calcium storage organelle in muscle cells that contains a calcium pump (SERCA) required for calcium reuptake and muscle relaxation. The activity of SERCA is regulated by the small integral-membrane subunits phospholamban (PLB) and sarcolipin (SLN). PLB is present in cardiac and smooth muscle, while SLN is found in skeletal muscle and the atria of the heart. Consequently, the regulatory mechanisms imposed by PLB and SLN have clinical implications for the treatment of heart disease. There is significant sequence homology in the transmembrane regions of PLB and SLN suggesting a similar mode of binding to SERCA; however, SLN has a unique and highly conserved C-terminal tail (27RSYQY) that is lacking in PLB. The structural differences in the luminal domains between these two proteins could be responsible for the subtle differences in their regulation of SERCA. We have functionally characterized alanine mutants of the C-terminal tail of SLN using co-reconstituted proteoliposomes that mimic the SR membrane. We found that Arg27 and Tyr31 are essential for SLN function. To further study the role of the luminal tail of SLN we also tested the effect of a truncated variant of SLN (Arg27stop) as well as chimeras of PLB consisting of the wild-type sequence with the five luminal residues of SLN added to its C-terminus. The Arg27stop form of SLN resulted in loss of function, while the PLBtail chimeras resulted in super-inhibition with characteristics reminiscent of the SERCA-PLB-SLN ternary complex. These functional results are being correlated with structural studies by cryo-electron microscopy of SERCA in complex with the PLBtail chimeras. Based on our results, we propose that SERCA inhibition by SLN is encoded in the C-terminal tail, and that the functional properties of SLN are transferred to PLB in the PLBtail chimeras.