DOI: 10.1073/pnas.1802417115
论文题名: Sodium and proton coupling in the conformational cycle of a MATE antiporter from Vibrio cholerae
作者: Claxton D.P. ; Jagessar K.L. ; Ryan Steed P. ; Stein R.A. ; Mchaourab H.S.
刊名: Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
出版年: 2018
卷: 115, 期: 27 起始页码: E6182
结束页码: E6190
语种: 英语
英文关键词: DEER
; EPR
; MATE
; NorM
; Transport mechanism
Scopus关键词: carboxylic acid
; carrier protein
; doxorubicin
; MATE protein
; proton
; sodium ion
; TM1 protein
; unclassified drug
; antiporter
; bacterial protein
; NorM protein, bacteria
; proton
; sodium
; amino acid sequence
; amino terminal sequence
; Article
; carboxy terminal sequence
; conformational transition
; controlled study
; crystal structure
; cytotoxicity
; double electron electron resonance spectroscopy
; nonhuman
; periplasm
; priority journal
; protein binding
; protein conformation
; protein domain
; site directed mutagenesis
; spectroscopy
; Vibrio cholerae
; chemistry
; genetics
; metabolism
; Vibrio cholerae
; Antiporters
; Bacterial Proteins
; Doxorubicin
; Protein Domains
; Protons
; Sodium
; Vibrio cholerae
英文摘要: Secondary active transporters belonging to the multidrug and toxic compound extrusion (MATE) family harness the potential energy of electrochemical ion gradients to export a broad spectrum of cytotoxic compounds, thus contributing to multidrug resistance. The current mechanistic understanding of ion-coupled substrate transport has been informed by a limited set of MATE transporter crystal structures from multiple organisms that capture a 12-transmembrane helix topology adopting similar outward-facing conformations. Although these structures mapped conserved residues important for function, the mechanistic role of these residues in shaping the conformational cycle has not been investigated. Here, we use double-electron electron resonance (DEER) spectroscopy to explore ligand-dependent conformational changes of NorM from Vibrio cholerae (NorM-Vc), a MATE transporter proposed to be coupled to both Na+ and H+ gradients. Distance measurements between spin labels on the periplasmic side of NorM-Vc identified unique structural intermediates induced by binding of Na+, H+, or the substrate doxorubicin. The Na+- and H+-dependent intermediates were associated with distinct conformations of TM1. Site-directed mutagenesis of conserved residues revealed that Na+- and H+-driven conformational changes are facilitated by a network of polar residues in the N-terminal domain cavity, whereas conserved carboxylates buried in the C-terminal domain are critical for stabilizing the drug-bound state. Interpreted in conjunction with doxorubicin binding of mutant NorM-Vc and cell toxicity assays, these results establish the role of ion-coupled conformational dynamics in the functional cycle and implicate H+ in the doxorubicin release mechanism. © 2018 National Academy of Sciences. All Rights Reserved. Citation statistics:
资源类型: 期刊论文
标识符: http://119.78.100.158/handle/2HF3EXSE/163697
Appears in Collections: 气候变化与战略
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作者单位: Claxton, D.P., Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232, United States; Jagessar, K.L., Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232, United States; Ryan Steed, P., Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232, United States; Stein, R.A., Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232, United States; Mchaourab, H.S., Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232, United States
Recommended Citation:
Claxton D.P.,Jagessar K.L.,Ryan Steed P.,et al. Sodium and proton coupling in the conformational cycle of a MATE antiporter from Vibrio cholerae[J]. Proceedings of the National Academy of Sciences of the United States of America,2018-01-01,115(27)