Cryo‐EM analysis provides new mechanistic insight into ATP binding to Ca ²⁺ ‐ATPase SERCA2b

Abstract

Sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) 2b is a ubiquitous SERCA family member that conducts Ca 2+ uptake from the cytosol to the ER. Herein, we present a 3.3 A resolution cryoelectron microscopy (cryo-EM) structure of human SERCA2b in the E1Á2Ca 2+ state, revealing a new conformation for Ca 2+ -bound SERCA2b with a much closer arrangement of cytosolic domains than in the previously reported crystal structure of Ca 2+ -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 E1Á2Ca 2+ state are located at similar positions to those in the E1Á2Ca 2+ -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 Ca 2+ . We propose a novel mechanism of ATP binding to SERCA2b.

Structural basis of the conformational and functional regulation of human SERCA2b, the ubiquitous endoplasmic reticulum calcium pump

“…[53] Using a protein-engineering approach, the N domain dynamics were restrained at a close distance to the A domain by introducing an interdomain disulfide bond between these two domains. [25] Nevertheless, significant autophosphorylation was observed in the E1 state,…”

“…[17,18] More recently, cryo-electron microscopy (EM) analysis of SERCA2b in the E1•2Ca 2+ state have provided another mechanistic insight into ATP entry. [24,25] It appears that even without a bound ATP molecule, Ca 2+ -bound SERCA proteins can adopt a compact cytosolic domain arrangement with a functional ATPbinding pocket; this closed state likely represents a state immediately prior to ATP binding. [24,25] Meanwhile, the cytosolic environment highly rich in ATP molecules tempts to speculate that the nucleotide may be bound to SERCA throughout its catalytic cycle.…”

“…[24,25] It appears that even without a bound ATP molecule, Ca 2+ -bound SERCA proteins can adopt a compact cytosolic domain arrangement with a functional ATPbinding pocket; this closed state likely represents a state immediately prior to ATP binding. [24,25] Meanwhile, the cytosolic environment highly rich in ATP molecules tempts to speculate that the nucleotide may be bound to SERCA throughout its catalytic cycle. [26,27] Given the submicomolar range of cytosolic Ca 2+ concentration, SERCA may likely exist predominantly as the E2•ATP (Ca 2+ -free but ATP-bound) form.…”

“…In line with this, our 3D classification based on a cryo-EM analysis of SERCA2b in the E1•2Ca 2+ state also generated an open conformationlike map, on which the crystal structure of SERCA1a in the same state (PDB ID:1SU4) can be roughly superimposed. [24,25] However, the reso- Given that the open conformation does not completely form a func-tional ATP-binding pocket (see also the section 'Mode of ATP binding by SERCA proteins'), it is possible that this conformation may represent an off-pathway state that can control the rate of ATP entry into the SERCA protein. [16,36] In this regard, a lifetime of the open conformation may be one of parameters that regulate the ATP-driven SERCA catalytic cycle.…”

“…[14,18] In support of this notion, our cryo-EM analysis of SERCA2b in the E1•2Ca 2+ state identified multiple closed conformations, all of which harbored an empty ATP-binding pocket with different sizes, likely due to the dynamics of the N domain. [25] Indeed, the N domain contains several flexible loops that interact with the A domain and can likely regulate the extent of compaction of the cytosolic domains in the E1 states. [53] Using a protein-engineering approach, the N domain dynamics were restrained at a close distance to the A domain by introducing an interdomain disulfide bond between these two domains.…”