Darobactin B stabilises a lateral-closed conformation of the BAM complex in E. coli cells

Samuel  F Haysom; Jonathan Machin; James  M Whitehouse; Jim  E Horne; Katherine Fenn; Yue Ma; Hassane El Mkami; Nils Böhringer; Till  F Schäberle; Neil  A Ranson; Sheena  E Radford; Christos Pliotas

doi:10.1002/anie.202218783

Darobactin B stabilises a lateral-closed conformation of the BAM complex in E. coli cells

Samuel F Haysom, Jonathan Machin, James M Whitehouse, Jim E Horne, Katherine Fenn, Yue Ma, Hassane El Mkami, Nils Böhringer, Till F Schäberle, Neil A Ranson, Sheena E Radford^*, Christos Pliotas^*

^*Corresponding author for this work

School of Physics and Astronomy

Research output: Contribution to journal › Article › peer-review

Abstract

The Β-barrel assembly machinery (BAM complex) is essential for outer membrane protein (OMP) folding in Gram-negative bacteria, and represents a promising antimicrobial target. Several conformational states of BAM have been reported, but all have been obtained under conditions which lack the unique features and complexity of the outer membrane (OM). Here, we use Pulsed Electron-Electron Double Resonance (PELDOR, or DEER) spectroscopy distance measurements to interrogate the conformational ensemble of the BAM complex in E. coli cells. We show that BAM adopts a broad ensemble of conformations in the OM, while in the presence of the antibiotic darobactin B (DAR-B), BAM's conformational equilibrium shifts to a restricted ensemble consistent with the lateral closed state. Our in-cell PELDOR findings are supported by new cryoEM structures of BAM in the presence and absence of DAR-B. This work demonstrates the utility of PELDOR to map conformational changes in BAM within its native cellular environment.

Original language	English
Article number	e202218783
Number of pages	8
Journal	Angewandte Chemie International Edition
Volume	62
Issue number	34
Early online date	31 May 2023
DOIs	https://doi.org/10.1002/anie.202218783
Publication status	Published - 21 Aug 2023

Keywords

PELDOR
BAM
Darobactin
In-cell EPR
CryoEM

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Haysom_2023_ACIE_Darobactin-B-Stabilises_CC
Copyright © 2023 The Authors. Angewandte Chemie published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Final published version, 594 KBLicence: CC BY

Cryogen-free arbitrary waveform EPR: Cryogen-free arbitrary waveform EPR for structural Biology and Biophysics
Bode, B. E. (PI), Lovett, J. E. (CoI), Pliotas, C. (CoI), Schwarz-Linek, U. (CoI), Smith, G. M. (CoI), Stewart, A. J. (CoI) & White, M. (CoI)
1/05/18 → 30/04/19
Project: Standard
State of the art pulse EPR instrumentati: State of the art pulse EPR instrumentation for long range distance measurements in biomacromolecules
Smith, G. M. (PI), Bode, B. E. (CoI), Naismith, J. (CoI), Schiemann, O. (CoI) & White, M. (CoI)
The Wellcome Trust
1/09/12 → 31/08/17
Project: Standard

Cite this

Haysom, S. F., Machin, J., Whitehouse, J. M., Horne, J. E., Fenn, K., Ma, Y., El Mkami, H., Böhringer, N., Schäberle, T. F., Ranson, N. A., Radford, S. E., & Pliotas, C. (2023). Darobactin B stabilises a lateral-closed conformation of the BAM complex in E. coli cells. Angewandte Chemie International Edition, 62(34), Article e202218783. https://doi.org/10.1002/anie.202218783

@article{3bfa81487ce549598eaee2bbc266b11f,

title = "Darobactin B stabilises a lateral-closed conformation of the BAM complex in E. coli cells",

abstract = "The Β-barrel assembly machinery (BAM complex) is essential for outer membrane protein (OMP) folding in Gram-negative bacteria, and represents a promising antimicrobial target. Several conformational states of BAM have been reported, but all have been obtained under conditions which lack the unique features and complexity of the outer membrane (OM). Here, we use Pulsed Electron-Electron Double Resonance (PELDOR, or DEER) spectroscopy distance measurements to interrogate the conformational ensemble of the BAM complex in E. coli cells. We show that BAM adopts a broad ensemble of conformations in the OM, while in the presence of the antibiotic darobactin B (DAR-B), BAM's conformational equilibrium shifts to a restricted ensemble consistent with the lateral closed state. Our in-cell PELDOR findings are supported by new cryoEM structures of BAM in the presence and absence of DAR-B. This work demonstrates the utility of PELDOR to map conformational changes in BAM within its native cellular environment.",

keywords = "PELDOR, BAM, Darobactin, In-cell EPR, CryoEM",

author = "Haysom, {Samuel F} and Jonathan Machin and Whitehouse, {James M} and Horne, {Jim E} and Katherine Fenn and Yue Ma and {El Mkami}, Hassane and Nils B{\"o}hringer and Sch{\"a}berle, {Till F} and Ranson, {Neil A} and Radford, {Sheena E} and Christos Pliotas",

note = "Funding: C.P. acknowledges the Biotechnology and Biological Sciences Research Council (BBSRC) (BB/S018069/1; BB/W019795/1) for funding. cwEPR data were collected at the Pliotas Laboratory (BB/S018069/1) and PELDOR data were collected at the University of St Andrews, supported by equipment funding from the Wellcome Trust (WT) (099149/Z/12/Z) and BBSRC (BB/R013780/1). CryoEM data were collected at the Astbury Biostructure Laboratory, funded by the University of Leeds and the WT (108466/Z/15/Z; 221524/Z/20/Z). J.E.H. and K.F. acknowledge funding from the Medical Research Council (MRC) (MR/P018491/1). S.F.H. is funded by the White Rose BBSRC DTP (BB/M011151/1). J.M. is funded by the WT (222373/Z/21/Z). SER holds a Royal Society Professorial Fellowship (RSRP/R1/211057). T.F.S and N.B. acknowledge funding from the German Federal Ministry of Education and Research (BMBF, via grant GBi2S and German Centrefor Infection Research (DZIF) 09.918).",

year = "2023",

month = aug,

day = "21",

doi = "10.1002/anie.202218783",

language = "English",

volume = "62",

journal = "Angewandte Chemie International Edition",

issn = "1433-7851",

publisher = "John Wiley & Sons, Ltd",

number = "34",

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TY - JOUR

T1 - Darobactin B stabilises a lateral-closed conformation of the BAM complex in E. coli cells

AU - Haysom, Samuel F

AU - Machin, Jonathan

AU - Whitehouse, James M

AU - Horne, Jim E

AU - Fenn, Katherine

AU - Ma, Yue

AU - El Mkami, Hassane

AU - Böhringer, Nils

AU - Schäberle, Till F

AU - Ranson, Neil A

AU - Radford, Sheena E

AU - Pliotas, Christos

N1 - Funding: C.P. acknowledges the Biotechnology and Biological Sciences Research Council (BBSRC) (BB/S018069/1; BB/W019795/1) for funding. cwEPR data were collected at the Pliotas Laboratory (BB/S018069/1) and PELDOR data were collected at the University of St Andrews, supported by equipment funding from the Wellcome Trust (WT) (099149/Z/12/Z) and BBSRC (BB/R013780/1). CryoEM data were collected at the Astbury Biostructure Laboratory, funded by the University of Leeds and the WT (108466/Z/15/Z; 221524/Z/20/Z). J.E.H. and K.F. acknowledge funding from the Medical Research Council (MRC) (MR/P018491/1). S.F.H. is funded by the White Rose BBSRC DTP (BB/M011151/1). J.M. is funded by the WT (222373/Z/21/Z). SER holds a Royal Society Professorial Fellowship (RSRP/R1/211057). T.F.S and N.B. acknowledge funding from the German Federal Ministry of Education and Research (BMBF, via grant GBi2S and German Centrefor Infection Research (DZIF) 09.918).

PY - 2023/8/21

Y1 - 2023/8/21

N2 - The Β-barrel assembly machinery (BAM complex) is essential for outer membrane protein (OMP) folding in Gram-negative bacteria, and represents a promising antimicrobial target. Several conformational states of BAM have been reported, but all have been obtained under conditions which lack the unique features and complexity of the outer membrane (OM). Here, we use Pulsed Electron-Electron Double Resonance (PELDOR, or DEER) spectroscopy distance measurements to interrogate the conformational ensemble of the BAM complex in E. coli cells. We show that BAM adopts a broad ensemble of conformations in the OM, while in the presence of the antibiotic darobactin B (DAR-B), BAM's conformational equilibrium shifts to a restricted ensemble consistent with the lateral closed state. Our in-cell PELDOR findings are supported by new cryoEM structures of BAM in the presence and absence of DAR-B. This work demonstrates the utility of PELDOR to map conformational changes in BAM within its native cellular environment.

AB - The Β-barrel assembly machinery (BAM complex) is essential for outer membrane protein (OMP) folding in Gram-negative bacteria, and represents a promising antimicrobial target. Several conformational states of BAM have been reported, but all have been obtained under conditions which lack the unique features and complexity of the outer membrane (OM). Here, we use Pulsed Electron-Electron Double Resonance (PELDOR, or DEER) spectroscopy distance measurements to interrogate the conformational ensemble of the BAM complex in E. coli cells. We show that BAM adopts a broad ensemble of conformations in the OM, while in the presence of the antibiotic darobactin B (DAR-B), BAM's conformational equilibrium shifts to a restricted ensemble consistent with the lateral closed state. Our in-cell PELDOR findings are supported by new cryoEM structures of BAM in the presence and absence of DAR-B. This work demonstrates the utility of PELDOR to map conformational changes in BAM within its native cellular environment.

KW - PELDOR

KW - BAM

KW - Darobactin

KW - In-cell EPR

KW - CryoEM

U2 - 10.1002/anie.202218783

DO - 10.1002/anie.202218783

M3 - Article

SN - 1433-7851

VL - 62

JO - Angewandte Chemie International Edition

JF - Angewandte Chemie International Edition

IS - 34

M1 - e202218783

ER -

Darobactin B stabilises a lateral-closed conformation of the BAM complex in E. coli cells

Abstract

Keywords

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Cryogen-free arbitrary waveform EPR: Cryogen-free arbitrary waveform EPR for structural Biology and Biophysics

State of the art pulse EPR instrumentati: State of the art pulse EPR instrumentation for long range distance measurements in biomacromolecules

Cite this