Nanosecond Fluorescence and Microsecond Simulation of SERCA Regulatory Interactions with Sarcolipin and Phospholamban

Abstract

We used subnanosecond-resolved fluorescence and all-atom molecular dynamics (MD) simulation to detect structural dynamics of the sarcoplasmic reticulum calcium/proton counter-transport ATPase (SERCA) and its inhibitory interactions with sarcolipin (SLN) and phospholamban (PLB). SLN and PLB individually regulate SERCA through direct interaction; SLN is highly expressed in fast-twitch muscle and atria, while PLB is highly expressed in slow-twitch muscle and ventricles. When SERCA, SLN, and PLB are expressed in the same human cell (vastus lateralis muscle, Takotsubo cardiomyopathy, ventricular mitral regurgitation), the three proteins form a “super-inhibitory” ternary complex (SERCA-SLN-PLB), whereby SERCA activity shows 5-fold decreased calcium affinity and 2-fold decreased maximal velocity. Forster resonance energy transfer (FRET) was used to quantify the complex equilibria of homo- and hetero-oligomeric interactions between SLN, PLB, and SERCA1a: five binary interactions were assayed and three assembly parameters were determined (binding affinity, oligomer number, interprobe distance). FRET results indicate that SLN and PLB show high-affinity self-association into homo-oligomers, and that SERCA forms heterodimers with SLN or PLB when co-expressed with either subunit individually, with SERCA having 3-fold higher affinity for SLN over PLB. To determine inhibition mechanisms of subunit-bound complexes, we used microsecond MD simulation of SERCA1a ± SLN or PLB in a POPC bilayer, with calcium, proton, or magnesium ions bound to transport sites of the SERCA transmembrane domain. MD results indicate that two potassium ions initially bind and pre-organize transport sites for calcium binding (SERCA activation), and that SLN and PLB individually inhibit SERCA by populating a calcium/potassium-free intermediate state with one bound proton at Glu771 (transport site I). We propose that super-inhibition of SERCA in the ternary complex is mediated by SLN/PLB-synergistic inhibition of proton/potassium exchange. Acknowledgments: this work was funded by grants to DDT (NIH-GM27906) and MEF (AHA-12SDG12060656).