Understanding the ABCs of multi drug resistance - tying the knot on the antibacterial peptide ABC transporter McjD

In recent years, outbreaks of E. coli infections in UK and Europe highlight the importance of understanding the disease mechanisms of such important pathogens and biomolecules. Bacteria have developed mechanisms to confer resistance to current antibiotics and one of the routes of resistance are membrane proteins that export drugs from the cell, called exporters. Membrane proteins represent around 30% of the proteomes of most organisms and more than 40% of drug targets and yet few structures of these molecules have been solved by X-ray crystallography. These proteins are usually embedded in oil like environment making them very difficult to work with. We first need to isolate them from the membrane using lipid mimics such as detergents. Membrane proteins usually have many functions and bacteria have developed mechanisms to utilise them in order to extrude antibiotics through these proteins. In order to develop new treatments it is important to understand the molecular mechanism of these important biological molecules. Using X-ray crystallography, we can gain a detailed understanding of the mechanism of these proteins; we need to grow crystals and expose them to X-rays in order to obtain the molecular structure. Certain drugs in the market are based on peptide chemistry and the bacteria have evolved these transporters to provide them with immunity by extruding them from inside the cell. One such family of proteins are the ATP-binding cassette transporters (ABC transporters) that are powered by the hydrolysis of ATP. Our group has recently solved the X-ray structure of an ABC exporter that is involved in antibacterial transport, McjD, which confers resistance to the antibacterial peptide MccJ25. We know that when the antibacterial peptide MccJ25 and ATP bind to McjD, the ABC exporter can adopt different conformations (a movement that mimics opening/closing of a gate) in order to extrude the toxic compound. In our crystal structure, McjD adopts a new conformation, nucleotide-bound outward occluded; the portion of the protein that sits in the membrane has a closed cavity where the MccJ25 can fit in. The structural information and biochemical experiments of this membrane protein will provide an important insight on the binding and recognition of antibacterial peptides and allow us to get a detailed picture of its mechanism. We aim to trap the transporter in different conformations and characterise it in the presence of the antibacterial peptides in order to reveal the mode of binding and transport. The biochemical experiments will try identifying (1) the binding and recognition of the peptide by McjD and (2) the conformational changes that McjD undergoes during peptide binding and transport (before ligand binding, occluded state and after the hydrolysis of ATP (resetting the system)). Understanding the structure and function of these important biological molecules will contribute to the understanding of the relationship between membrane protein and substrate recognition and provide valuable information to structural biology and pharmacology.

The overall aim of this proposal is to gain a detailed insight in the molecular mechanism of antibacterial peptide and multi drug exporters. This study will also allows us to gain a very detailed picture on the ligand recognition by ABC exporters. ABC exporters are powered by the hydrolysis of ATP and transport their substrate via the alternating access mechanism with a twist. There is little structural information on this important class of exporters. We have selected the novel antibacterial peptide ABC exporter McjD to expand our knowledge and we have solved the structure of McjD at 2.7 Å resolution. The structure is in a novel conformation for ABC exporters, nucleotide bound outward occluded. The structure represents an intermediate between outward- and inward-facing states. We propose that the initial driving force from an outward to inward state is proton driven rather than ATP dependent. We have reconstituted McjD in proteoliposomes and it retains its ATPase activity; its ligand, MccJ25, can induce the ATPase activity. We want to study how McjD recognises its substrate by NMR. In light of the new conformation we want to further characterise the new state by mutagenesis, PELDOR and X-ray crystallography. We have identified residues in the transmembrane region that form salt-bridges and we propose to disrupt them and measure their ability to transport its ligand in proteoliposomes. We want to investigate the conformational changes associated with ATP hydrolysis. We have already obtained initial crystals and the structure shows that upon ATP hydrolysis the two nucleotide binding domains have separated; unexpectedly the transmembrane domain is still in an occluded state. We want to optimise these crystals in order to build a reliable model for the post-hydrolysis state. We will also combine PELDOR measurements in bicelles to measure specific distances and compare them to the crystal structure.

Many of the antibiotics in the market disrupt bacterial cellular functions, but bacteria have found ways to utilise membrane proteins, efflux pumps and transporters, to expel these compounds and detoxify the cells. The objective of this proposal is the elucidation of the detailed mechanism of the antibacterial peptide ABC transporter McjD. There is still limited structural information on membrane proteins and there is a need to expand our current knowledge of membrane protein structure and function.The beneficiaries will be the national and international academic community, healthcare, industry sector and education sectors. The immediate impacts will be in the academic community and in particular the membrane protein biology field (structural biology, molecular dynamics, functional characterisation). Bacterial ABC exporters are involved in multi drug resistance and the new structures and ligand binding studies will provide the community with a more detailed understanding of ABC exporters' mechanism and expand our knowledge on this important family of proteins.
The PDRA will be trained on handling membrane proteins (expression, purification and crystallisation) and develop lab skills that they can transfer to their next post, either academic or industrial environment. Many labs work with membrane proteins, and their experience with membrane proteins will put the PDRA in a very strong position for future job applications. The PI has also been involved in the organisation of workshops for sharing their expertise on membrane proteins in the past. The meeting brought together experts from the membrane protein field to share their expertise with PhD and post doctoral staff.
We will raise awareness to other scientists of our findings through high impact peer-reviewed journals and presentation of our work at international conferences. Media will be involved for the promotion of the findings to other scientists as well as the general public. Both Diamond Light Source and the Imperial College media teams through interviews, podcasts and annual reports have been involved in promoting our research to the public in the past. The Beis lab has been actively involved in engaging with local school students in promoting science.
The medium to long-term impact will involve the pharmaceutical and healthcare industry. These exporters are involved in multidrug resistance and their structure can be exploited from the pharmaceutical industry in the development of novel inhibitors that cannot be transported by these ABC exporters (rather than direct inhibition of the transporters). The structure can provide useful information on the binding and transport of the antibacterial peptides that in turn can help design new drugs for fighting bacterial infections. In long term, the general public will benefit from the development and release of any new drugs or therapies from pharmaceutical companies that can tackle multidrug resistance. The 2011 outbreak of Escherichia coli infection in Europe resulted in nearly 4,000 infected people and 50 deaths. Therefore, development of new antibacterials is essential.