An octameric PqiC toroid stabilises the outer-membrane interaction of the PqiABC transport system

Benjamin F Cooper; Giedrė Ratkevičiūtė; Luke A Clifton; Hannah Johnston; Rachel Holyfield; David J Hardy; Simon G Caulton; William Chatterton; Pooja Sridhar; Peter Wotherspoon; Gareth W Hughes; Stephen Cl Hall; Andrew L Lovering; Timothy J Knowles

doi:10.1038/s44319-023-00014-4

An octameric PqiC toroid stabilises the outer-membrane interaction of the PqiABC transport system

Benjamin F Cooper, Giedrė Ratkevičiūtė, Luke A Clifton, Hannah Johnston, Rachel Holyfield, David J Hardy, Simon G Caulton, William Chatterton, Pooja Sridhar, Peter Wotherspoon, Gareth W Hughes, Stephen Cl Hall, Andrew L Lovering, Timothy J Knowles^*

^*Corresponding author for this work

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Abstract

The E. coli Paraquat Inducible (Pqi) Pathway is a putative Gram-negative phospholipid transport system. The pathway comprises three components: an integral inner membrane protein (PqiA), a periplasmic spanning MCE family protein (PqiB) and an outer membrane lipoprotein (PqiC). Interactions between all complex components, including stoichiometry, remain uncharacterised; nevertheless, once assembled into their quaternary complex, the trio of Pqi proteins are anticipated to provide a continuous channel between the inner and outer membranes of diderms. Here, we present X-ray structures of both the native and a truncated, soluble construct of the PqiC lipoprotein, providing insight into its biological assembly, and utilise neutron reflectometry to characterise the nature of the PqiB-PqiC-membrane interaction. Finally, we employ phenotypic complementation assays to probe specific PqiC residues, which imply the interaction between PqiB and PqiC is less intimate than previously anticipated.

Original language	English
Pages (from-to)	82-101
Number of pages	20
Journal	EMBO Reports
Volume	25
Issue number	1
DOIs	https://doi.org/10.1038/s44319-023-00014-4
Publication status	Published - 12 Jan 2024

Bibliographical note

Acknowledgements:
We wish to thank the funding bodies who support us, BFC, GR, HJ, RH and PW were/are jointly funded by the BBSRC and the University of Birmingham (through the Midlands Integrative Biosciences Training Partnership - Grant No.BB/M01116X/1). DJH, GWH and TJK were supported by BBSRC Research Grant No. BB/S017283/1. We thank the Science and Technology Facilities Council, the ISIS Neutron & Muon Source for allocation of beamtime (RB2300004 and RB2220703) and Diamond Light Source for access to beamline I04 under proposal number mx26803.

© 2024. The Author(s).

Keywords

Lipid Transport
Outer Membrane Biogenesis
Gram NegativeCell Envelope
Pqi
Envelope Spanning

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CooperB2024octamericFinal published version, 18.4 MBLicence: Creative Commons: Attribution (CC BY)

Crossing the periplasmic void, elucidating the mechanisms of phospholid transport in Gram-negative bacteria
Hughes, G. (Co-Investigator), Knowles, T. (Principal Investigator) & Lovering, A. (Co-Investigator)
Biotechnology & Biological Sciences Research Council
1/09/19 → 30/04/23
Project: Research

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Cooper, B. F., Ratkevičiūtė, G., Clifton, L. A., Johnston, H., Holyfield, R., Hardy, D. J., Caulton, S. G., Chatterton, W., Sridhar, P., Wotherspoon, P., Hughes, G. W., Hall, S. C., Lovering, A. L., & Knowles, T. J. (2024). An octameric PqiC toroid stabilises the outer-membrane interaction of the PqiABC transport system. EMBO Reports, 25(1), 82-101. https://doi.org/10.1038/s44319-023-00014-4

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abstract = "The E. coli Paraquat Inducible (Pqi) Pathway is a putative Gram-negative phospholipid transport system. The pathway comprises three components: an integral inner membrane protein (PqiA), a periplasmic spanning MCE family protein (PqiB) and an outer membrane lipoprotein (PqiC). Interactions between all complex components, including stoichiometry, remain uncharacterised; nevertheless, once assembled into their quaternary complex, the trio of Pqi proteins are anticipated to provide a continuous channel between the inner and outer membranes of diderms. Here, we present X-ray structures of both the native and a truncated, soluble construct of the PqiC lipoprotein, providing insight into its biological assembly, and utilise neutron reflectometry to characterise the nature of the PqiB-PqiC-membrane interaction. Finally, we employ phenotypic complementation assays to probe specific PqiC residues, which imply the interaction between PqiB and PqiC is less intimate than previously anticipated.",

keywords = "Lipid Transport, Outer Membrane Biogenesis, Gram NegativeCell Envelope, Pqi, Envelope Spanning",

author = "Cooper, {Benjamin F} and Giedrė Ratkevi{\v c}iūtė and Clifton, {Luke A} and Hannah Johnston and Rachel Holyfield and Hardy, {David J} and Caulton, {Simon G} and William Chatterton and Pooja Sridhar and Peter Wotherspoon and Hughes, {Gareth W} and Hall, {Stephen Cl} and Lovering, {Andrew L} and Knowles, {Timothy J}",

note = "Acknowledgements: We wish to thank the funding bodies who support us, BFC, GR, HJ, RH and PW were/are jointly funded by the BBSRC and the University of Birmingham (through the Midlands Integrative Biosciences Training Partnership - Grant No.BB/M01116X/1). DJH, GWH and TJK were supported by BBSRC Research Grant No. BB/S017283/1. We thank the Science and Technology Facilities Council, the ISIS Neutron & Muon Source for allocation of beamtime (RB2300004 and RB2220703) and Diamond Light Source for access to beamline I04 under proposal number mx26803. {\textcopyright} 2024. The Author(s).",

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Cooper, BF, Ratkevičiūtė, G, Clifton, LA, Johnston, H, Holyfield, R, Hardy, DJ, Caulton, SG, Chatterton, W, Sridhar, P, Wotherspoon, P, Hughes, GW, Hall, SC, Lovering, AL & Knowles, TJ 2024, 'An octameric PqiC toroid stabilises the outer-membrane interaction of the PqiABC transport system', EMBO Reports, vol. 25, no. 1, pp. 82-101. https://doi.org/10.1038/s44319-023-00014-4

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T1 - An octameric PqiC toroid stabilises the outer-membrane interaction of the PqiABC transport system

AU - Cooper, Benjamin F

AU - Ratkevičiūtė, Giedrė

AU - Clifton, Luke A

AU - Johnston, Hannah

AU - Holyfield, Rachel

AU - Hardy, David J

AU - Caulton, Simon G

AU - Chatterton, William

AU - Sridhar, Pooja

AU - Wotherspoon, Peter

AU - Hughes, Gareth W

AU - Hall, Stephen Cl

AU - Lovering, Andrew L

AU - Knowles, Timothy J

N1 - Acknowledgements: We wish to thank the funding bodies who support us, BFC, GR, HJ, RH and PW were/are jointly funded by the BBSRC and the University of Birmingham (through the Midlands Integrative Biosciences Training Partnership - Grant No.BB/M01116X/1). DJH, GWH and TJK were supported by BBSRC Research Grant No. BB/S017283/1. We thank the Science and Technology Facilities Council, the ISIS Neutron & Muon Source for allocation of beamtime (RB2300004 and RB2220703) and Diamond Light Source for access to beamline I04 under proposal number mx26803. © 2024. The Author(s).

PY - 2024/1/12

Y1 - 2024/1/12

N2 - The E. coli Paraquat Inducible (Pqi) Pathway is a putative Gram-negative phospholipid transport system. The pathway comprises three components: an integral inner membrane protein (PqiA), a periplasmic spanning MCE family protein (PqiB) and an outer membrane lipoprotein (PqiC). Interactions between all complex components, including stoichiometry, remain uncharacterised; nevertheless, once assembled into their quaternary complex, the trio of Pqi proteins are anticipated to provide a continuous channel between the inner and outer membranes of diderms. Here, we present X-ray structures of both the native and a truncated, soluble construct of the PqiC lipoprotein, providing insight into its biological assembly, and utilise neutron reflectometry to characterise the nature of the PqiB-PqiC-membrane interaction. Finally, we employ phenotypic complementation assays to probe specific PqiC residues, which imply the interaction between PqiB and PqiC is less intimate than previously anticipated.

AB - The E. coli Paraquat Inducible (Pqi) Pathway is a putative Gram-negative phospholipid transport system. The pathway comprises three components: an integral inner membrane protein (PqiA), a periplasmic spanning MCE family protein (PqiB) and an outer membrane lipoprotein (PqiC). Interactions between all complex components, including stoichiometry, remain uncharacterised; nevertheless, once assembled into their quaternary complex, the trio of Pqi proteins are anticipated to provide a continuous channel between the inner and outer membranes of diderms. Here, we present X-ray structures of both the native and a truncated, soluble construct of the PqiC lipoprotein, providing insight into its biological assembly, and utilise neutron reflectometry to characterise the nature of the PqiB-PqiC-membrane interaction. Finally, we employ phenotypic complementation assays to probe specific PqiC residues, which imply the interaction between PqiB and PqiC is less intimate than previously anticipated.

KW - Lipid Transport

KW - Outer Membrane Biogenesis

KW - Gram NegativeCell Envelope

KW - Pqi

KW - Envelope Spanning

U2 - 10.1038/s44319-023-00014-4

DO - 10.1038/s44319-023-00014-4

M3 - Article

C2 - 38228789

SN - 1469-221X

VL - 25

SP - 82

EP - 101

JO - EMBO Reports

JF - EMBO Reports

IS - 1

ER -

An octameric PqiC toroid stabilises the outer-membrane interaction of the PqiABC transport system

Abstract

Bibliographical note

Keywords

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Projects

Crossing the periplasmic void, elucidating the mechanisms of phospholid transport in Gram-negative bacteria

Cite this