Solid-State NMR Structures of Phospholamban or Sarcolipin Bound to Calcium ATPase (SERCA) Reveal the Mode of Inhibition

Abstract

Sarco(endo)plasmic reticulum calcium ATPase (SERCA) transports Ca2+ ions against a concentration gradient from cytoplasm to sarcoplasmic reticulum, initiating muscle relaxation. SERCA activity is regulated by two single-span membrane proteins: phospholamban (PLN) or sarcolipin (SLN). Although the recent crystal structures offered a prime view of these complexes (SERCA/SLN and SERCA/PLN), the mechanism of regulation is still unknown. Here, we used a combination of oriented and magic angle spinning (MAS) solid-state NMR spectroscopy to gain atomistic level insights into the mechanism of regulation of SERCA by both PLN and SLN in fully hydrated lipid bilayer membranes.To map protein-protein interactions within lipid membranes, we designed innovative labeling strategies involving various combinations of isotopic labeling schemes, paramagnetic probes, and thioalkylation of SERCA's cysteines. The restraints obtained from the experimental data were used to model the complexes which were further refined using restrained molecular dynamics simulations in explicit lipid bilayers. The structures of SERCA in complex with PLN or SLN provide a detailed view of the binding interface and shed light on the mechanism of SERCA regulation (i.e., inhibition and activation). Knowledge of the biophysical basis of SERCA regulation offers unprecedented opportunity for the rational design of the mutants for gene therapy. We expect the approach and methods used by us to be generally applicable for mapping protein-protein interactions in membrane protein complexes larger than 100 kDa, which are typically inaccessible to other experimental methods.