@article{3388974879544619875d29b0ed33bca7,
title = " Structural Basis of Sarco/Endoplasmic Reticulum Ca 2+ -ATPase 2b Regulation via Transmembrane Helix Interplay ",
abstract = " Sarco/endoplasmic reticulum (ER) Ca 2+ -ATPase 2b (SERCA2b) is a ubiquitously expressed membrane protein that facilitates Ca 2+ uptake from the cytosol to the ER. SERCA2b includes a characteristic 11 th transmembrane helix (TM11) followed by a luminal tail, but the structural basis of SERCA regulation by these C-terminal segments remains unclear. Here, we determined the crystal structures of SERCA2b and its C-terminal splicing variant SERCA2a, both in the E1-2Ca 2+ -adenylyl methylenediphosphonate (AMPPCP) state. Despite discrepancies with the previously reported structural model of SERCA2b, TM11 was found to be located adjacent to TM10 and to interact weakly with a part of the L8/9 loop and the N-terminal end of TM10, thereby inhibiting the SERCA2b catalytic cycle. Accordingly, mutational disruption of the interactions between TM11 and its neighboring residues caused SERCA2b to display SERCA2a-like ATPase activity. We propose that TM11 serves as a key modulator of SERCA2b activity by fine-tuning the intramolecular interactions with other transmembrane regions. ",
keywords = "Ca -ATPase, Ca homeostasis, X-ray crystallography, endoplasmic reticulum, membrane protein",
author = "Michio Inoue and Nanami Sakuta and Satoshi Watanabe and Yuxia Zhang and Kunihito Yoshikaie and Yoshiki Tanaka and Ryo Ushioda and Yukinari Kato and Junichi Takagi and Tomoya Tsukazaki and Kazuhiro Nagata and Kenji Inaba",
note = "Funding Information: We thank the beamline scientists of SPring-8 and Photon Factory for their assistance with X-ray diffraction data collection. This work was supported by CREST, JST (to K.I. and K.N., grant number JPMJCR13M6 ), Grants-in-Aid for Scientific Research on Innovative Areas from MEXT ( 15H04335 and 26116005 to K.I. and 26119007 to T.T.), the Takeda Science Foundation (to K.I.), and the Platform Project for Supporting Drug Discovery and Life Science Research/Platform for Drug Discovery, Informatics, and Structural Life Science (PDIS) from the Japan Agency for Medical Research and Development (AMED) (to K.I.). This work was also supported by Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research [BINDS]) from AMED ( JP18am0101075 and JP18am0101078 to J.T. and Y.K.). Funding Information: We thank the beamline scientists of SPring-8 and Photon Factory for their assistance with X-ray diffraction data collection. This work was supported by CREST, JST (to K.I. and K.N. grant number JPMJCR13M6), Grants-in-Aid for Scientific Research on Innovative Areas from MEXT (15H04335 and 26116005 to K.I. and 26119007 to T.T.), the Takeda Science Foundation (to K.I.), and the Platform Project for Supporting Drug Discovery and Life Science Research/Platform for Drug Discovery, Informatics, and Structural Life Science (PDIS) from the Japan Agency for Medical Research and Development (AMED) (to K.I.). This work was also supported by Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research [BINDS]) from AMED (JP18am0101075 and JP18am0101078 to J.T. and Y.K.). M.I. constructed a large-scale expression system for SERCA2b and SERCA2a and established the purification methods. M.I. and J.T. prepared the vector for the overexpression of PA-tagged SERCA2b. N.S. purified SERCA2a on a large scale. M.I. N.S. K.Y. Y.T. and T.T. crystallized SERCA2a and SERCA2b using the LCP method. M.I. N.S. and S.W. collected and analyzed X-ray diffraction data and determined the structures of SERCA2a and SERCA2b. S.W. merged the diffraction data and refined the structures. M.I. and Y.Z. constructed plasmids for expressing SERCA2b mutants; prepared microsomal SERCA2b WT, F1019/V1028 mutants, and SERCA2a WT; and measured their ATPase activities. Y.K. prepared anti-PA antibody and conjugated Sepharose beads. K.I. R.U. and K.N. designed the study. K.I. supervised the work. M.I. and S.W. prepared the figures. K.I. M.I. and S.W. wrote the manuscript. All of the authors discussed the results and approved the manuscript. The authors declare no competing interests. Publisher Copyright: {\textcopyright} 2019 The Author(s)",
year = "2019",
month = apr,
day = "23",
doi = "10.1016/j.celrep.2019.03.106",
language = "English",
volume = "27",
pages = "1221--1230.e3",
journal = "Cell Reports",
issn = "2211-1247",
publisher = "Cell Press",
number = "4",
}