@article{4fb6c1ba4bb44a6eadcb6602368d9639,
title = "Multiple sub-state structures of SERCA2b reveal conformational overlap at transition steps during the catalytic cycle",
abstract = "Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pumps Ca2+ into the endoplasmic reticulum (ER). Herein, we present cryo-electron microscopy (EM) structures of three intermediates of SERCA2b: Ca2+-bound phosphorylated (E1P·2Ca2+) and Ca2+-unbound dephosphorylated (E2·Pi) intermediates and another between the E2P and E2·Pi states. Our cryo-EM analysis demonstrates that the E1P·2Ca2+ state exists in low abundance and preferentially transitions to an E2P-like structure by releasing Ca2+ and that the Ca2+ release gate subsequently undergoes stepwise closure during the dephosphorylation processes. Importantly, each intermediate adopts multiple sub-state structures including those like the next one in the catalytic series, indicating conformational overlap at transition steps, as further substantiated by atomistic molecular dynamic simulations of SERCA2b in a lipid bilayer. The present findings provide insight into how enzymes accelerate catalytic cycles.",
keywords = "CP: Molecular biology, SERCA, SERCA2b, calcium, cryo-EM single-particle analysis, endoplasmic reticulum, molecular dynamics simulation",
author = "Yuxia Zhang and Chigusa Kobayashi and Xiaohan Cai and Satoshi Watanabe and Akihisa Tsutsumi and Masahide Kikkawa and Yuji Sugita and Kenji Inaba",
note = "Funding Information: This work was supported by funding from AMED-CREST (21gm1410006h0001) to K.I. JSPS KAKENHI to K.I. (18H03978, 21H04758, and 21H05247) and Y.Z. (21K15036), funding from the MITUBISHI Foundation to K.I. and the Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS) from the Japan Agency for Medical Research and Development (AMED) under grant number JP19am0101115 (support number: 1025). Computational studies were supported by JSPS KAKENHI grants to Y.S. (19H05645 and 21H05249) and Program for Promoting Research on the Supercomputer Fugaku (Biomolecular dynamics in a living cell, JPMXP1020200101). Computational resources were provided to Y.S. via use of the Fugaku supercomputer through the RIKEN Center for Computational Science (ra000003), HPCI (hp210172 and hp210177), and RIKEN HOKUSAI BigWaterfall. Y.Z. performed almost all experiments, structure modeling, and structure refinement. A.T. and M.K. performed acquisition of cryo-EM images. C.K. and Y.S. performed molecular dynamics simulations. S.W. assisted in structure modeling and refinement. Y.Z. and K.I. prepared figures and wrote the manuscript. All authors discussed the results, critically read the manuscript, and approved the manuscript for submission. K.I. supervised this work. The authors declare no competing interests. Funding Information: This work was supported by funding from AMED-CREST ( 21gm1410006h0001 ) to K.I., JSPS KAKENHI to K.I. ( 18H03978 , 21H04758 , and 21H05247 ) and Y.Z. ( 21K15036 ), funding from the MITUBISHI Foundation to K.I., and the Basis for Supporting Innovative Drug Discovery and Life Science Research ( BINDS ) from the Japan Agency for Medical Research and Development (AMED) under grant number JP19am0101115 (support number: 1025). Computational studies were supported by JSPS KAKENHI grants to Y.S. ( 19H05645 and 21H05249 ) and Program for Promoting Research on the Supercomputer Fugaku (Biomolecular dynamics in a living cell, JPMXP1020200101 ). Computational resources were provided to Y.S. via use of the Fugaku supercomputer through the RIKEN Center for Computational Science (ra000003), HPCI (hp210172 and hp210177), and RIKEN HOKUSAI BigWaterfall. Publisher Copyright: {\textcopyright} 2022 The Authors",
year = "2022",
month = dec,
day = "6",
doi = "10.1016/j.celrep.2022.111760",
language = "English",
volume = "41",
journal = "Cell Reports",
issn = "2211-1247",
publisher = "Cell Press",
number = "10",
}