Lipopolysaccharide translocation across the Gram-negative inner membrane; development and exploitation of reagents and knowledge to guide early stage

Lead Research Organisation: University of Dundee
Department Name: School of Life Sciences

Abstract

Lipopolysaccharide (LPS) is a key component of the highly protective cell surface of Gram-negative pathogens. LPS is produced and displayed on the cell surface by the actions of a series of enzymes and a multi-protein transport system termed the Lpt complex. Bacterial survival is compromised if LPS production is curtailed and genetic studies reveal that many of the genes encoding proteins involved in LPS production/transport are essential. A high level of research activity has concentrated on characterisation of the cytosolic biosynthetic enzymes and the proteins that involve transfer from periplasm to cell surface, at the outer membrane. Our plan is to focus on the missing area, and to characterise those proteins involved in the transfer of components across the inner membrane. The three key proteins to be investigated are MsbA, LptF and LptG. MsbA is an ATPase driven flippase, which acts in conjunction with thepotentially heterodimeric LptF-LptG pair. It is an ATP-binding cassette transporter and the fold is known. However, for LptF-G there is are no known orthologous structures. The LptF and LptG proteins typically share about 20% sequence identity and secondary structure predictions suggest a common fold, predicted to comprise 6 transmembrane helices, is present. These two proteins may form homo or heterodimers, a feature we aim to clarify at the onset of the project.

Publications

10 25 50
 
Description Yersinia pestis is a Gram-negative bacterium, which causes plague. In 1996, two drug-resistant strains of plague were isolated from Madagascar. These strains have acquired resistance to most of the drugs currently used for therapy and prophylaxis of plague (strains 16/95 and 17/95). Outbreaks of plague have happened in Asia and Africa over the last few decades and the plague is now recognised as a re-emerging disease. Moreover, there are evidence suggesting that modified strains of plague were developed by the biological warfare agents (BWA) programs of several countries and therefore there are serious concerns associated with the possibility of Y. pestis being used as a BWA.

One of the main aims of this project was to prepare a foundation for developing novel therapeutic agents against Yersinia pestis. The project was focusing on the proteins involved in the biogenesis of cell wall in Y. pestis - Penicillin Binding Proteins (PBPs). Two proteins - YpPBP2 and YpPBP3 were identified as essential in this organism based on Transposon Directed Insertion Sequencing Data (TRADIS) provided by defence science and technology laboratory (Dstl) as well as knockout and homology data.

In the course of this project structures of YpPBP2 and YpPBP3 were solved using X-ray crystallography in apo and in complex with several known Beta-lactams. Details revealing composition of catalytic domains of YpPBP2 and YpPBP3 were acquired for the first time. Data about protein-ligand interactions can now be used to improve current therapeutic agents targeting PBPs and to develop novel compounds. A fluorescence-based assay was developed to simultaneously validate the binding of multiple Beta-lactams to YpPBP2 and YpPBP3. Out of 17 modern Beta-lactams tested six compounds were capable of inhibiting both YpPBP2 and YpPBP3. A set of compounds was identified using several biochemical techniques as possible binders of Y. pestis - these compounds could be later used as a starting points for developing novel ligands targeting YpPBP2 and YpPBP3.

Protocol for producing pure and homogeneous sample of bifunctional protein responsible for cell wall biogenesis of Y. pestis - PBP1B was also achieved. This information can now be used for determining structure of YpPBP1B.
Exploitation Route Detailed information about the protein-ligand interactions in YpPBP3 & YpPBP2 can be used to develop novel ligands against these proteins to target cell wall biogenesis in Y. pestis and other Gram-negative organisms. Protocols developed for achieving recombinant expression of these proteins as well as YpPBP1B can be used for performing other biochemical assays on these proteins and to study protein-protein interactions.
Sectors Aerospace, Defence and Marine,Healthcare,Government, Democracy and Justice,Pharmaceuticals and Medical Biotechnology,Security and Diplomacy

URL https://journals.iucr.org/f/services/forthcoming.html
 
Title Biolayer interferometry 
Description Bio-layer interferometry (BLI) is a label-free technology for measuring biomolecular interactions. It is an optical analytical technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilised protein on the biosensor tip, and an internal reference layer. Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real-time. The binding between a ligand immobilized on the biosensor tip surface and an analyte in solution produces an increase in optical thickness at the biosensor tip, which results in a wavelength shift, which is a direct measure of the change in thickness of the biological layer. Interactions are measured in real time, providing the ability to monitor binding specificity, rates of association and dissociation, or concentration, with precision and accuracy. 
Type Of Material Technology assay or reagent 
Provided To Others? Yes  
Impact Biolayer interferometry is used in my research to study interaction between proteins of interest and ligands. This technique can also be exploited to identify novel ligands interactiong with the protein of interest. 
URL https://www.fortebio.com/interactions/Spring_2012/page5.html
 
Title Immobilized metal affinity chromatography (IMAC) 
Description Immobilized metal affinity chromatography (IMAC) is a specialized variant of affinity chromatography where the proteins or peptides are separated according to their affinity for metal ions that have been immobilized by chelation to an insoluble matrix. At pH values around neutral, the amino acids histidine, tryptophan, and cysteine form complexes with the chelated metal ions (e.g., Zn2+, Cu2+, Cd2+, Hg2+, Co2+, Ni2+, and Fe2+). They can then be eluted by reducing the pH, increasing the mobile phase ionic strength, or adding ethylenediaminetetraacetic acid (0.05 M) to the mobile phase. This technique is especially suited for purifying recombinant proteins as poly-histidine fusions and for membrane proteins and protein aggregates where detergents or high-ionic-strength buffers are required. 
Type Of Material Biological samples 
Provided To Others? Yes  
Impact I frequently use immobilized metal affinity chromatography as a first step in protein purification. It allows producing a relatively clean protein sample just in one purification step. This sample, however normally is not suitable for crystallisation and requires subsequent purification steps e.g. size exclusion chromatography. 
URL https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2909483/
 
Title Size-exclusion chromatography 
Description Size exclusion chromatography is a method where separation of different compounds occurs according to their size measured by how efficiently they penetrate the pores of the stationary phase. Size exclusion is the only mode of chromatography that is not intended to involve binding of proteins to the resin. The pore structure of the resin provides a molecular sieve, where smaller molecules can access the entire volume of the pores and large molecules are excluded from the pores. If a mixture of proteins differing in size is applied to a size exclusion column, the largest proteins will emerge first and smallest last. Molecules above a certain size do not penetrate the pores at all. Normally, these are the first to elute from the column; the volume at which they elute is termed the "excluded volume." 
Type Of Material Biological samples 
Provided To Others? Yes  
Impact Size-exclusion chromatography is a non-denaturing protein purification technique, which is frequently used in our laboratory for producing pure and homogeneous samples suitable for X-ray crystallography. Not only this technique can be used for protein purification, it can also help to assess the quaternary structure of the protein. 
URL https://pubs.acs.org/doi/abs/10.1021/a19600193
 
Title Thermal shift assay (differential scanning fluorimetry) 
Description A thermal shift assay quantifies the change in thermal denaturation temperature of a protein under varying conditions. The differing conditions that can be examined are very diverse, include: pH, salts, additives, drugs, drug leads etc. The binding of ligands can increase the thermal stability of a protein. Thermostable proteins are often more useful than their non-thermostable counterparts. Protein crystallisation is more successful for proteins with a higher melting point and adding buffer components that stabilise proteins improve the likelihood of protein crystals forming. 
Type Of Material Technology assay or reagent 
Provided To Others? Yes  
Impact Differential scanning fluorimetry (DSF) is widely used in our lab. On multiple occasions this method helped to alleviate issues with protein solubility and allowed preparation of the protein samples suitable for crystallisation. DSF has also helped to identify the ligands, which provided an increase in the melting temperatures of the proteins of interest. These ligands were then successfully used for co-crystallisation experiments. 
URL https://www.nature.com/articles/nprot.2007.321
 
Description Defence Science and Technology Laboratory (DSTL) 
Organisation Defence Science & Technology Laboratory (DSTL)
Country United Kingdom 
Sector Public 
PI Contribution Quaternary reports are made to DSTL outlining progress made in exploration of bacterial proteins that can be targeted for developing novel therapeutic strategies.
Collaborator Contribution DSTL provided us with some crucial information about the importance of certain bacterial proteins and indicated what proteins could be explored for developing novel therapeutics agains G-ve pathogens. DSTL also partially provides funding for my research.
Impact As an outcome of the collaboration with DSTL, the following was done: - Refinement of crystal structure and subsequent publication: "Open and compressed conformations of Francisella tularensis ClpP" together with Laura Díaz-Sáez and William N. Hunter. - Deposition of Crystal structure of an ABC transporter related protein from Burkholderia pseudomallei in Protein Data Bank (accession code: 6FLX, publication will follow). - Crystal structure of Penicillin Binding Protein 3 from Yersinia pestis was solved in apo form and in complex with carbenicillin (currently awaits deposition).
Start Year 2015
 
Description MRC Festival open day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact In the course of MRC Festival I was showing X-ray facility and biochemistry labs to the members of local communities and pupils from local schools. I was explaining the principles of the structure-based drug design, X-ray crystallography and protein-ligand interactions as well as describing the impact provided by research conducted in the University of Dundee. Some of the pupils were particularly interested and asked many questions - I believe several of them might pursue a scientific career in future.
Year(s) Of Engagement Activity 2018
URL https://www.ppu.mrc.ac.uk/open-day
 
Description University of dundee open doors day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Around 60-70 school kids and several members of public attended the Life Science building of the university of Dundee on the Open Doors event. Visitors were had an opportunity to have a look at the X-ray facilities/crystallisation room and laboratories we have. They were introduced to the principles of X-ray crystallography and familiarised with protein composition, folding and functions. They were also shown how 3D models of the protein could be used to design a drug targeting the protein. Several kids showed high interest to the provided information and mentioned that they want to do a Life Sciences degree after finishing school.
Year(s) Of Engagement Activity 2016,2017