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Molecular mechanisms of multidrug binding by the bacterial ABC transporter Sav1866

Reference	BB/F008333/1
Principal Investigator / Supervisor	Professor Hendrik Willem van Veen
Co-Investigators / Co-Supervisors
Institution	University of Cambridge
Department	Pharmacology
Funding type	Research
Value (£)	301,448
Status	Completed
Type	Research Grant
Start date	12/05/2008
End date	11/05/2011
Duration	36 months

Abstract

Multidrug transporters mediate the extrusion of structurally unrelated drugs from prokaryotic and eukaryotic cells. As a result of this efflux activity, the cytoplasmic drug concentration in the cell is lowered to subtoxic levels, and, hence, cells become multidrug resistant. Multidrug transporters interfere with the chemotherapeutic treatment of cancers and infections by pathogenic microorganisms. This research proposal focuses on the membrane transporter Sav1866, a bacterial homologue of the human multidrug resistance P-glycoprotein, for which recently a high-resolution crystal structure was determined [Nature 443: 180-185 (2007)]. Sav1866 is an ATP-binding cassette (ABC) transporter containing one membrane domain with 6 transmembrane spanning segments and one nucleotide-binding domain. Similar to its homologue LmrA from Lactococcus lactis [EMBO J. 19: 2503-2514 (2000)] and MsbA from Escherichia coli [J. Biol. Chem. 278:35193-35198], Sav1866 is most likely active as a homodimer. In our recent functional studies on MsbA and Sav1866, we have identified regions in the membrane domains where change-in-specificity mutations, observed for P-glycoprotein [Trends Pharmacol. Sci. 27:195-203], might co-localise. In this project, we will study drug-protein interactions in Sav1866 is detail. In particular, we aim: (i) to identify locations of drug-binding surfaces in Sav1866 by cysteine crosslinking and by site-specific proteolytic cleavage, (ii) to use molecular docking techniques to predict drug-protein interactions, and (iii) to study structure-activity relationships in drug-binding surfaces and drug molecules. This strategy will add important new information about the molecular mechanisms by which this unique class of multidrug transporters recognizes its substrates, and may lead to the development of new drugs that could bypass or inhibit drug efflux in a clinical setting.

Summary

Multidrug transporters are very small molecular machines that are present at the surface of human cells and microorganisms. These ingenious machines provide the cell with a defense mechanism against toxic compounds which are harmful to them. The activity of multidrug transporters interferes with the drug-based control, also termed 'chemotherapy', of human tumours and infectious pathogenic microorganisms, in which drugs are designed to kill the cell. Multidrug transporters bind structurally unrelated drugs and toxins at the inside of the cell, and then move these compounds to the outside of the cell, away from the targets with which the drugs are supposed to interact. Hence, multidrug transporters act as drug pumps that are able to lower the drug concentration inside the cell to levels at which these drugs are inactive and useless. As drugs now have lost their therapeutic effects, the cell has become 'multidrug resistant'. This project focuses on the bacterial multidrug transporter Sav1866, which is very similar to one of the major multidrug pumps in human. Sav1866 is made by the pathogenic bacterium Staphylococcus aureus, but we use the non-pathogenic, food-grade bacterium Lactococcus lactis to study the properties of the pump. Sav1866 is particularly interesting as researchers have very recently been able to determine its three-dimensional structure. In this project, we study how Sav1866 recognizes and binds drugs. This knowledge might allow us to rationally design new drugs that can poison or circumvent the activity of these transport proteins in microorganisms and human cells to improve chemotherapy of cancers and infectious diseases.

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	Microbiology, Pharmaceuticals, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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