Characterizing Protein Binding of Phospholamban to SERCA using Chemical Cross-Linking

Abstract

Phospholamban (PLB) is a small integral membrane phosphoprotein that inhibits the activity of the sarcoplasmic reticulum Ca-ATPase (SERCA) by reducing its affinity for Ca. Several groups have shown that site-specific chemical cross-linking of PLB to SERCA is abolished by elevated Ca, suggesting the complex is dissociated by Ca-binding. However we and others have previously observed that Ca does not abolish fluorescence resonance energy transfer (FRET) from SERCA to PLB, suggesting that PLB remains bound in high Ca. One possible explanation for the apparently conflicting results is that SERCA transition from E2 (Ca-free) to E1 (Ca-bound) results in translocation of PLB from the canonical binding site to a novel site. This translocation could diminish site-specific cross-linking without abolishing FRET. In support of this hypothesis, preliminary computational docking experiments suggest a possible secondary PLB binding site on SERCA on the outside of helix M9. This site is analogous to the FXYD protein binding site determined for the related P-type ATPase, the Na/K-ATPase. To investigate PLB-SERCA binding to the putative novel site, we labeled PLB with a photoactivatable cross-linker, benzophenone-4-maleimide and quantified cross-linking with SDS-PAGE and immunoblotting. We observed a high molecular weight species reactive to PLB and SERCA antibodies, both in the absence and presence of Ca in cardiac myocytes and a heterologous expression system. Overall, the data suggest that PLB and SERCA do not dissociate at high Ca. Planned experiments will investigate the location and functional significance of the proposed novel PLB binding site on SERCA.