Biotechnology and Biological Sciences Research Council (BBSRC)
BB/M011151/1
英国
Wellcome Trust
222373/Z/21/Z
英国
Wellcome Trust
105220/Z/14/Z
英国
Research Foundation - Flanders (FWO)
G0G0818N
ベルギー
Royal Society
RSRP/R1/211057
英国
引用
ジャーナル: Nat Commun / 年: 2021 タイトル: The role of membrane destabilisation and protein dynamics in BAM catalysed OMP folding. 著者: Paul White / Samuel F Haysom / Matthew G Iadanza / Anna J Higgins / Jonathan M Machin / James M Whitehouse / Jim E Horne / Bob Schiffrin / Charlotte Carpenter-Platt / Antonio N Calabrese / ...著者: Paul White / Samuel F Haysom / Matthew G Iadanza / Anna J Higgins / Jonathan M Machin / James M Whitehouse / Jim E Horne / Bob Schiffrin / Charlotte Carpenter-Platt / Antonio N Calabrese / Kelly M Storek / Steven T Rutherford / David J Brockwell / Neil A Ranson / Sheena E Radford / 要旨: The folding of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the β-barrel assembly machinery (BAM). How lateral opening in the β-barrel of the major subunit ...The folding of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the β-barrel assembly machinery (BAM). How lateral opening in the β-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.
名称: lid-locked beta-barrel assembly machinery (BAM) complex (BamABCDE) bound by a bactericidal Fab fragment タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: all
7NBX (lateral-open BAM-LL cryoEM structure) and 7BM5 (Fab1 crystal structure) were rigid fitted into the electron density in UCSF Chimera. One round of molecular dynamics based flexible fitting was then set up in VMD and run in NAMD to fit the structures into the BAM-LL-Fab1 density. The resulting structure was finally real-space refined in phenix to generate the final model.
精密化
空間: REAL / プロトコル: FLEXIBLE FIT / 温度因子: 183
得られたモデル
PDB-7ncs: Lateral-open conformation of the lid-locked BAM complex (BamA E435C S665C, BamBDCE) bound by a bactericidal Fab fragment