regulation of membrane invagination / cell envelope / bacteriocin transport / protein import / cell division site / transmembrane transporter activity / protein transport / cell cycle / cell division / membrane / plasma membrane 類似検索 - 分子機能
Tol-Pal system, TolQ / Tol-Pal system protein TolR / Biopolymer transport protein ExbD/TolR / Biopolymer transport protein ExbD/TolR / MotA/TolQ/ExbB proton channel / MotA/TolQ/ExbB proton channel family 類似検索 - ドメイン・相同性
Tol-Pal system protein TolQ / Tol-Pal system protein TolR 類似検索 - 構成要素
Biotechnology and Biological Sciences Research Council (BBSRC)
BB/V008056/1
英国
European Research Council (ERC)
742555
European Union
引用
ジャーナル: Proc Natl Acad Sci U S A / 年: 2023 タイトル: Tunable force transduction through the cell envelope. 著者: Daniel P Williams-Jones / Melissa N Webby / Cara E Press / Jan M Gradon / Sophie R Armstrong / Joanna Szczepaniak / Colin Kleanthous / 要旨: The outer membrane (OM) of Gram-negative bacteria is not energised and so processes requiring a driving force must connect to energy-transduction systems in the inner membrane (IM). Tol (Tol-Pal) and ...The outer membrane (OM) of Gram-negative bacteria is not energised and so processes requiring a driving force must connect to energy-transduction systems in the inner membrane (IM). Tol (Tol-Pal) and Ton are related, proton motive force- (PMF-) coupled assemblies that stabilise the OM and import essential nutrients, respectively. Both rely on proton-harvesting IM motor (stator) complexes, which are homologues of the flagellar stator unit Mot, to transduce force to the OM through elongated IM force transducer proteins, TolA and TonB, respectively. How PMF-driven motors in the IM generate mechanical work at the OM via force transducers is unknown. Here, using cryoelectron microscopy, we report the 4.3Å structure of the TolQR motor complex. The structure reaffirms the 5:2 stoichiometry seen in Ton and Mot and, with motor subunits related to each other by 10 to 16° rotation, supports rotary motion as the default for these complexes. We probed the mechanism of force transduction to the OM through in vivo assays of chimeric TolA/TonB proteins where sections of their structurally divergent, periplasm-spanning domains were swapped or replaced by an intrinsically disordered sequence. We find that TolA mutants exhibit a spectrum of force output, which is reflected in their respective abilities to both stabilise the OM and import cytotoxic colicins across the OM. Our studies demonstrate that structural rigidity of force transducer proteins, rather than any particular structural form, drives the efficient conversion of PMF-driven rotary motions of 5:2 motor complexes into physiologically relevant force at the OM.
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 281 K / 装置: FEI VITROBOT MARK IV / 詳細: Blot force 10 Blot time 3 sec Wait time 2 sec.
詳細
sample purified by affinity chromatography (His-tagged TolR) and SEC before application to grids in 50 mM Tris/HCl pH 7.5, 300 mM NaCl, 2 mM EDTA, 0.01% LMNG
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電子顕微鏡法
顕微鏡
FEI TITAN KRIOS
撮影
フィルム・検出器のモデル: GATAN K3 (6k x 4k) / 平均電子線量: 46.39 e/Å2