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Molecular mechanisms of multidrug transporters: from bacteria to man

 

Dr Hendrik W. van Veen - Group leader

E-mail: hwv20@cam.ac.uk

Tel: +44 1223 765295/334073
Fax: +44 1223 334100

 

Keywords

Research tackling antimicrobial resistance (AMR), inhibition of antibiotic resistance mechanisms (ARM), Clare College, drug resistance, antibiotic resistance, anticancer drug resistance, membrane transporters, multidrug transporters, drug efflux, molecular basis of drug recognition, mechanisms of transport, bioenergetics, inhibitors, electrophysiological & biochemical/biophysical assays.

 

Investigator biography

Hendrik van Veen earned his B.Sc. and M.Sc. degrees in Biochemistry and PhD degree in Microbiology (supervisors Profs. Alex Zehnder and Wil Konings) cum laude at Wageningen University, the Netherlands. In 1996, his thesis on solute transport in bacteria was awarded the triennial Kluyver Award of the Netherlands Society of Microbiology. After post-doctoral work at the Universities of Groningen (Prof. Wil Konings) and Oxford (Prof. Chris Higgins), he received a research fellowship from the Royal Netherlands Academy of Arts and Sciences (KNAW) to work on bacterial multidrug resistance at Groningen University. During this time, he obtained a Max Gruber Award in Biochemistry from the University of Groningen. In 2001 he became associated with the University of Cambridge, Department of Pharmacology, first as a University Lecturer and University Senior Lecturer, and currently as Reader in Molecular Pharmacology. He is a teaching fellow at Clare College, and leader of a research group aiming at the mechanisms of antibiotic and anticancer drug recognition and transport by multidrug transporters in pro- and eukaryotic cells. He serves on various editorial boards, and committees of funding agencies and societies. Click here for a citation overview of his publications, and here for a curriculum vitae.

 

Group contributions

We biochemically and functionally characterized the first-known bacterial homologue of the human multidrug resistance P-glycoprotein and expressed this ABC transporter (LmrA) in mammalian cells for a direct comparison of its pharmacological properties with those of P-glycoprotein. Established a role for ion-coupling in the energetics of LmrA in studies in proteoliposomes and by electrophysiological techniques, and demonstrated for the first time that catalytic reactions in LmrA can mediate ATP synthesis in the presence of reverse substrate gradients. Determined drug-protein stoichiometries using equilibrium drug binding and transport assays. Established the transport of small molecule drugs by the lipid-A ABC transporter MsbA and a role for proton coupling in the activity of this transporter. Identified structural elements in the human breast cancer resistance protein (ABCG2) that are responsible for protein-drug interactions in the substrate binding pocket. Identified the presence of multiple drug interaction sites in a MFS multidrug transporter (LmrP) and demonstrated variable (substrate-dependent) proton coupling. Discovered that ion coupling in certain bacterial MATE transporters is based on the simultaneous input of the proton-motive force and sodium-motive force. Generated mechanistic interpretations of crystal structures and cryo-EM structures of multidrug transporters in ABC and RND families.

 

Lab members

 

Our expertise

We have expertise in the elucidation of structure-function relationships in multidrug transporters that underlie their ability to be polyspecific and mediate metabolic energy-dependent substrate extrusion. Membrane transporters are overexpressed and characterized using expression systems ranging from bacteria, yeast and insect cells to mammalian cells. The interaction of transporters and a variety of ligands (drugs, bile salts, inorganic ions, nucleotides) are analysed in transport measurements and equilibrium binding assays, and by studying association and dissociation kinetics of radioligand binding. Substrate binding is also assessed by fluorescence anisotropy and measurements of intrinsic protein and tryptophan fluorescence, as well as other biophysical methods. Protein-ligand and protein-protein interactions are deduced from ATPase and nucleotide-binding activities and from photoaffinity labelling and protein cross-linking using chemical and thiol-reactive cross-linkers and substrate analogues. Transport (flux) measurements are carried out in intact cells, membrane vesicles, and proteoliposomes containing purified and functionally reconstituted proteins. We use electrophysiological techniques to study electrogenic substrate transport reactions by purified and functionally reconstituted membrane transporters. In addition to the biochemical, molecular biological and electrophysiological techniques, we apply crystallographic, NMR, EPR, and mass spectrometry techniques on proteins of interest in direct collaborations with others. Hence, we integrate a wide range of techniques in studies on the mechanisms of membrane transporters.

 

Selected publications

Van Veen, H.W., Venema, K., Bolhuis, B., Oussenko, I., Kok, J., Poolman, B., Driessen, A.J.M., and Konings, W.N. (1996) Multidrug resistance mediated by a bacterial homolog of the human drug transporter MDR1. Proc. Natl. Acad. Sci. USA 93: 10668-10672.

Van Veen, H.W., Callaghan, R., Soceneantu, L., Sardini, A., Konings, W.N., and Higgins, C.F. (1998) A bacterial antibiotic-resistance gene that complements the human multidrug-resistance P-glycoprotein gene. Nature 391: 291-295.

Van Veen, H.W., Margolles, A., Müller, M., Higgins, C.F., and Konings, W.N. (2000) The ATP-binding cassette transporter LmrA mediates multidrug transport by an alternating two-site (two-cylinder engine) mechanism. EMBO J. 19: 2503-2514.

Venter, H., Shilling, R., Velamakanni, S., Balakrishnan, L., and Van Veen, H.W. (2003) An ABC transporter with a secondary-active multidrug translocator domain. Nature 426: 866-869.

Reuter, G., Janvilisri, T., Venter, H., Shahi, S., Balakrishnan, L., and Van Veen, H.W. (2003) The ATP-binding cassette multidrug transporter LmrA and lipid transporter MsbA have overlapping substrate specificities. J. Biol. Chem 278: 35193-35198.

Woebking, B., Velamakanni, S., Federici, L., Seeger, M.A., Murakami, S., and Van Veen, H.W. (2008) Functional role of transmembrane helix 6 in drug binding and transport by the ABC transporter MsbA. Biochemistry 47: 10904-10914.

Barrera, N.P., Isaacson, S.C., Zhou, M., Bavro, V.N., Welch, A., Schaedler, T.A., Seeger, M.A., Miguel, R.N., Korkhov, V.M., van Veen, H.W., Venter, H., Walmsley, A.R., Tate, C.G., Robinson, C.V. (2009) Mass spectrometry of membrane transporters reveals subunit stoichiometry and interactions. Nature Methods 6(8):585-587.

Van Veen, H.W. (2010) Structural biology: Last of the multidrug transporters. Nature 467: 926-927.

Schaedler, T.A., and Van Veen, H.W. (2010) A flexible cation binding site in the multidrug major facilitator superfamily transporter LmrP is associated with variable proton coupling. FASEB J. 24: 3653-3661.

Doshi, R., Ali, A., Shi, W., Freeman, E.V., Fagg, L.A., van Veen, H.W. (2013) Molecular disruption of the power stroke in the ATP-binding cassette transport protein MsbA. J Biol Chem. 288(10):6801-6813.

Choudhury*, H.G., Tong*, Z., Mathavan, I., Li, Y., Iwata, S., Zirah, S., Rebuffat, S., Van Veen, H.W., and Beis, K. (2014) Structure of an antibacterial peptide ATP-binding cassette transporter in a novel outward occluded state. Proc. Natl. Acad. Sci. USA. 111: 9145-9150. (* shared first authors)

Jin, Y., Nair, A., and Van Veen, H.W. (2014) Multidrug transport protein NorM from Vibrio cholerae simultaneously couples to sodium- and proton-motive force. J. Biol. Chem. 289(21): 14624-14632

Singh, H., Velamakanni, S., Deery, M.J., Howard, J., Wei, S.L., and Van Veen, H.W. (2016) ATP-dependent substrate transport by the ABC transporter MsbA is proton-coupled. Nature Commun. 7, 12387 doi: 10.1038/ncomms12387.

Fitzpatrick, A. W. P., Llabrés, S., Neuberger, A., Blaza, J.N., Bai, X.C., Okada, U., Murakami, S., van Veen, H.W., Zachariae, U., Scheres, S.H.W., Luisi, B.F., Du, D. (2017) Structure of the MacAB-TolC ABC-type tripartite multidrug efflux pump. Nature Microbiol. 2:17070. doi: 10.1038/nmicrobiol.2017.70.

Okada, U., Yamashita, E., Neuberger, A., Morimoto, M., van Veen, H.W., Murakami, S. (2017) Crystal structure of tripartite-type ABC transporter MacB from Acinetobacter baumannii. Nature Commun. 8(1):1336.   doi: 10.1038/ s41467-017-01399-2.

Du, D., Wang-Kan, X., Neuberger, A., van Veen, H.W., Pos, K.M., Piddock, L.J.V., Luisi, B.F. (2018) Multidrug efflux pumps: structure, function and regulation. Nature Reviews Microbiol. doi: 10.1038/s41579-018-0048-6.

Agboh, K., Lau, C.H.F., Khoo, Y.S.K., Singh, H., Raturi, S., Nair, A.V., Howard, J., Chiapello, M., Feret, R., Deery, M.J., Murakami, S., van Veen, H.W. (2018) Powering the ABC multidrug exporter LmrA: How nucleotides embrace the ion-motive force. Science Advances 4, eaas9365.