Cryo-EM analysis provides new mechanistic insight into ATP binding to Ca²⁺-ATPase SERCA2b

Sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) 2b is a ubiquitous SERCA family member that conducts Ca²⁺ uptake from the cytosol to the ER. Herein, we present a 3.3 Å resolution cryo-electron microscopy (cryo-EM) structure of human SERCA2b in the E1·2Ca²⁺ state, revealing a new conformation for Ca²⁺-bound SERCA2b with a much closer arrangement of cytosolic domains than in the previously reported crystal structure of Ca²⁺-bound SERCA1a. Multiple conformations generated by 3D classification of cryo-EM maps reflect the intrinsically dynamic nature of the cytosolic domains in this state. Notably, ATP binding residues of SERCA2b in the E1·2Ca²⁺ state are located at similar positions to those in the E1·2Ca²⁺-ATP state; hence, the cryo-EM structure likely represents a preformed state immediately prior to ATP binding. Consistently, a SERCA2b mutant with an interdomain disulfide bridge that locks the closed cytosolic domain arrangement displayed significant autophosphorylation activity in the presence of Ca²⁺. We propose a novel mechanism of ATP binding to SERCA2b.