Supercharging the calcium pump: Identification of an activation hotspot on SERCA by cryo-EM

The sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) is a ubiquitous P-type ATPase that restores cytosolic Ca 2+ to the sarco-endoplasmic reticulum. SERCA is essential for cardiac Ca 2+ cycling and cellular energy metabolism. Several small molecules enhance SERCA function and show promise in models of metabolic and cardiovascular diseases. However, the structural basis for SERCA activation has remained unknown, hindering mechanism-driven lead optimization. Here we present cryo-EM structures of SERCA bound to two chemically distinct activators: the quinoline derivative CDN1163 (2.6 Å resolution) and a benzofuran derivative UM-52 (3.1 Å resolution). Biochemical assays show that both compounds stimulate Ca 2+ -dependent ATPase activity of SERCA without altering the apparent Ca²⁺ affinity. The structures reveal a previously unrecognized “activation hotspot” in the transmembrane domain, a shallow groove formed by helices M3 and M4 and capped by M1. Despite low chemical similarity, both activators occupy the same pocket and share conserved interactions with Ser 265 , Trp 272 , and Phe 296 . These residues are unique to SERCA and help explain selectivity relative to other P-type ATPases. Activator binding stabilizes a catalytically competent conformation, shifting SERCA toward an E1-like state poised for ATP binding and coordinated movements of the M1-M4 bundle and the cytosolic domains. Notably, density consistent with a detergent acyl chain bridges an otherwise open cavity adjacent to the compound, suggesting that altered protein-lipid interactions may contribute to activation. Together, these findings define a structural framework for SERCA activation and provide a blueprint for rational design of next-generation SERCA activators. SIGNIFICANCE STATEMENT SERCA pumps Ca²⁺ into the sarco-endoplasmic reticulum, enabling muscle relaxation and shaping calcium signals across tissues. Small-molecule SERCA activators improve cardiac and metabolic phenotypes in animal models, but drug development has been limited by the absence of a defined binding site and activation mechanism. We determined cryo-EM structures of SERCA bound to two distinct activators, CDN1163 and UM-52. Both compounds occupy a groove formed by transmembrane segments M3-M4, anchored by a hydrogen bond to the SERCA-specific Ser 265 and aromatic contacts near Trp 272 and Phe 296 . An acyl chain bridges a gap in the binding pocket toward M1, suggesting that protein-lipid coupling may play a role in SERCA activation. These results directly enable structure-mechanism guided design of next-generation selective SERCA activators.