Molecular Basis for Substrate Recognition of Outer Membrane Proteins of the Human Pathogen Pseudomonas Aeruginosa

Lead Research Organisation: University of Southampton
Department Name: School of Chemistry


Bacterial membranes are notoriously impermeable - this is one of the reasons they are so difficult to beat with currently available antibiotics. This is leading to the widespread development of so-called 'superbugs'; bacteria that are resistant to many different antibiotics. This phenomenon provides modern healthcare with a serious problem. Pseudomonas aeruginosa is a human, animal and plant pathogen that is resistant to many different antibiotics. In humans, it tends to target those with already compromised immune systems, and is responsible for a large proportion of hospital acquired infections.

Before we can embark on rational drug design to combat Pseudomonas aeruginosa, we must understand the routes via which the bacterial cell can be penetrated. Given that nature has had the benefit of years of evolution, it makes sense to exploit this by first trying to understand how molecules necessary for the survival of the bacterium, enter it. In this project we will use a range of computational methods supported by electrophysiology experiments by our collaborators, to elucidate the molecular pathways taken by dipeptides to enter the cell through the OprD protein. This protein is one member of the largest family of substrate-specific channel proteins located within the outer membrane of Pseudomonas aeruginosa.

Recently reported studies reveal that using a combination of antibiotics (combination therapy) can be more effective than administering just one type. However the molecular-level consequences of combination therapy are currently completely unknown. Thus, in addition to studying OprD under 'normal' conditions, we will also explore how the protein behaves when its local environment is altered by polymyxin B1- an antibiotic.

Technical Summary

The development of bacterial resistance to antibiotics for humans, animals and plants is a major problem for modern healthcare. This has recently been highlighted by a World Health Organization report, 2014 (ISBN: 978 92 4 156474 8) which states: "resistance to common bacteria has reached alarming levels in many parts of the world and that in some settings, few, if any, of the available treatment options remain effective for common infections"

The overall aim is to facilitate the rational design of novel antibiotics. To do this, we must lay the molecular-level groundwork. This project aims to do just that by focussing on the Gram-negative bacterium, Pseudomonas aeruginosa. We will employ a combination of molecular modelling, docking, steered molecular dynamics, umbrella sampling and straightforward equilibrium molecular dynamics to elucidate the molecular recognition pathways utilised by dipeptides as they permeate through the protein, OprD to get across the outer membrane of Pseudomonas aeruginosa. The combination of methods we plan to use will provide us with information about individual molecular interactions that mediate the protein-substrate recognition and subsequent permeation processes including quantitative information regarding the relative energetics involved. Thus this project will constitute one of the most detailed computational studies of a bacterial outer membrane protein.

Our calculations will be backed up by experimental data from electrophysiology studies by our colleagues in Bremen. The bi-directional flow of information between theory and experiment will serve to improve the theoretical models and also guide future experimental studies.

Planned Impact

The science represented by this proposal will have an impact on the academic and private and public sectors, facilitated by our plans for dissemination, outreach and public engagement.

Public sector:
The impact on the public sector will largely be in the context of education. We have recently started a new initiative to improve visualisation of academic posters to render them more interactive and hence ideal for educational purposes. This is initiative is supported by RIM, who are providing Blackberry hardware for SK to produce prototype posters than incorporate touch screen technology for interactive visualisation of simulation data. This project will provide data for such educational posters. Furthermore we propose to fully develop our prototype 'arcade machine' for running interactive molecular dynamics simulations for 'hands-on' demonstrations. We will visit schools to give talks and demonstrations throughout the duration of the project.

Commercial sector:
There is scope for commercialisation of the interactive posters as well as the software for searching the database of simulation movies. Novel antibiotics will of course have an impact for the commercial sector, and we plan to work closely with pharmaceutical companies to realise this impact as efficiently as possible. Realistically, the development of new drugs will require computational and experimental work beyond the current project.

Staff working on this project will develop skills in: a range of cutting-edge molecular simulation methodologies, including docking, free-energy calculations, steered molecular dynamics simulations as well as experience of working with complex bacterial membrane models. We are one of only 2 or 3 laboratories in the world with this expertise, thus this represents an excellent training opportunity for the postdoc. The postdoc will be required to interact with experimental colleagues and therefore will learn valuable communication, collaboration and interpretation skills.

In summary, the results of our proposed project will underpin the development of novel antibiotics, in particular those that are potent against Pseudomonas aeruginosa, thus the project will contribute to enhancing the quality of life and health. Commercialisation of the antibiotics will improve the economic competitiveness of the UK. The realistic timescales to achieving the full impact of the project will be a few years after its completion given we propose to first establish the fundamental principles of protein-substrate recognition, it will take time for these to be translated into viable therapeutics. Thus this project represents impact that will be realised in the long-term and will be long-lasting.


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Boags A (2017) Progress in Molecular Dynamics Simulations of Gram-Negative Bacterial Cell Envelopes. in The journal of physical chemistry letters

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Im W (2020) Molecular Simulations of Gram-Negative Bacterial Membranes Come of Age. in Annual review of physical chemistry

Description We have discovered how the various components of the outer membrane of Gram-negative bacteria move relative to each other. We have shown that the outer leaflet diffuses 10 x slower than the inner leaflet. We have discovered that the outer membrane of Gram-negative bacteria becomes easier to bend when proteins are added to it, and the converse is true for the inner membrane.

We have shown how arginine permeates through the protein OprD and how the LPS of the surrounding membrane helps to guide the substrate to the protein.

We have shown that proteins binding to the cell wall in a non-covalent manner play a key role in maintaining the position of the cell wall and also its conformation. In regions of protein depletion, the cell wall becomes buckled.
Exploitation Route These findings can be taken forward to develop novel drugs with enhanced uptake by bacterial cells. New experiments can be designed to test our predictions regarding membrane curvature.
Sectors Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology

Description Combining quantum and classical methods to study bacterial membrane enzymes
Amount £126,931 (GBP)
Organisation The Leverhulme Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2018 
End 07/2021
Description Collaboration with Ben Luisi 
Organisation University of Cambridge
Department Department of Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution We have performed molecular dynamics simulations of cryo-EM structures of E.coli efflux pumps, determined by Ben Luisi and his team. We have identified lipid sorting behaviour of the proteins and therefore we have added value to their structural data
Collaborator Contribution They provided the structural data which we used to initiate our simulations.
Impact J Phys Chem Lett. 2017 Nov 16;8(22):5513-5518. doi: 10.1021/acs.jpclett.7b02432. Epub 2017 Oct 31. It Is Complicated: Curvature, Diffusion, and Lipid Sorting within the Two Membranes of Escherichia coli. Hsu PC1, Samsudin F1, Shearer J1, Khalid S1.
Start Year 2016
Description Collaboration with Colin Kleanthous & Hagan Bayley (Oxford) 
Organisation University of Oxford
Department Division of Cardiovascular Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution We are using molecular dynamics simulations to understand the experimental observations of Colin K and Hagan B, namely that some peptides will only translocate through the poplin OmpF from one direction.
Collaborator Contribution Experimental data on peptide translocation through OmpF
Impact Paper in prep
Start Year 2017
Description Collaboration with David Roper, Warwick 
Organisation University of Warwick
Department Biological Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We have combined molecular dynamics simulations (from our side) with structural biology data (from David Roper's laboratory) to provide unprecedented insights into the structure-function relationships of the jaw subdomain of peptidoglycan glycosyltransferases for lipid II polymerization
Collaborator Contribution They have provided us with an X-ray structure of a protein.
Impact A manuscript is about to be submitted most likely to Embo J
Start Year 2017
Description Collaboration with Professor Steve Matthews 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution This is a collaboration based on understanding a range of protein bacterial membrane proteins. We have provided models of the outer membranes of E.coli in which the proteins are found and also parameters for adding the lipidated portions of those proteins for which these regions must be added to the X-ray structure (in the first instance this has been CsgG).
Collaborator Contribution Professor Steve Matthews and Dr Sarah Rouse have provided optimised structures of proteins and equilibrated nanodisc models. Together we have simulated a number of these proteins. Furthermore they have provided NMR data related to the structure of the proteins.
Impact One paper so far: This is a multidisciplinary project with the project partners contributing NMR data, mutational biophysical data, and computational models. We have contributed computational models and expertise.
Start Year 2018
Title PyCGTool 
Description PyCGTool enables parameterisation of coarse-grain molecules, systematically from atomistic trajectories. The paper describing the first version of the software is available here: The software has since been updated and is now further being improved to add procedures for automated mapping of the coarse-grain models from their atomistic equivalents. 
Type Of Technology Software 
Year Produced 2017 
Open Source License? Yes  
Impact The software has been used by several groups to parameterise new molecules (e.g. cyclodextrins). We have used it to parameterise lipopolysaccharide molecules. 
Description Advanced technology to support research, innovation and economic growth in the UK 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Reform hosted a policy roundtable on the opportunities for advanced technology in the UK in May 2019, with the kind support of Hewlett Packard Enterprise. The session was introduced by Chris Skidmore MP, then Minister of State for Universities, Science, Research and Innovation, and Professor Mark Parsons, Director at the Edinburgh Parallel Computing Centre.

The Minister stressed the importance of international collaboration in R&D, reaffirming the UK's ambition to strengthen and enrich existing
partnerships, as well as to develop new global partnerships - as outlined in the then recently announced International Research & Innovation
Strategy. The discussion also focused on the opportunities the upcoming Comprehensive Spending Review would offer to the science, research and innovation sectors, and on the need to build a strong case for. investment in emerging technologies - including quantum technology, performance computing (HPC) and robotics.

During this meeting it was highlighted that High-performance computing is considered a game-changing technology, which will be fundamental
to the UK's ability to maintain its global competitiveness in the science, research and innovation sectors.
Year(s) Of Engagement Activity 2019
Description Hamied Foundation UK-India Antimicrobial Resistance Meeting 2019 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I was part of an expert panel of scientists and medics from the UK and India assembled to discuss how we can (a) combat existing antimicrobial resistance and (b) prevent or slow down the development of resistance in future. The chief medical officer of the UK was also in attendance and a gave talk. Collaborations between scientists from both countries were established and existing ones were strengthened. An emerging theme was that all levels must be addressed, from atoms and molecules through to patients, communities and the environment. I helped establish this theme.
Year(s) Of Engagement Activity 2019
Description Newton-Bhabha workshop on developing new antibiotics 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact The Royal Society of Chemistry, Public Health England and some Indian organisations held a joint event in Bangalore, India for UK and Indian scientists in academia and industry to discuss potential future collaborations in the area of tackling the development of antimicrobial resistance to antibiotics. A number of collaborations were set up, and new grant applications were planned. As I direct consequence of this visit, I have been invited to give talks at Surrey University and Birmingham University in the UK, and in Delhi in India. The participants of the meeting agreed that we would change the way we deal with research data, and make it more accessible for each other.
Year(s) Of Engagement Activity 2017
Description Pint of Science talk: Making movies of Bacterial membranes 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact I gave a talk in a pub to an audience consisting of members of the general public who had bought tickets to the event. The talk explained some of the science behind antimicrobial resistance followed by some details of my own work in terms of 'making movies' of the bacterial membranes so we can understand how they protect bacteria. I talked about how we have to understand the membranes in molecular detail if we are to design new drugs with the ability to permeate across these membranes. Audience members said they had a much better understanding of how they must not abuse antibiotics, after the talk.
Year(s) Of Engagement Activity 2017