mycobacterial lipids; X-ray crystallography; Multidrug efflux pumps; protein reconstitution; ABC transporter; virulence factors; Mycobacterium smegmatis; secondary-active transporters; Structure-based biochemistry; Membrane proteins; Mycobacterium tuberculosis; pathogenic bacteria; Lactococcus lactis
Hürlimann Lea M., Hohl Michael, Seeger Markus A. (2017), Split tasks of asymmetric nucleotide‐binding sites in the heterodimeric ABC exporter EfrCD, in The FEBS Journal
, 284(11), 1672-1687.
Timachi M Hadi, Hutter Cedric AJ, Hohl Michael, Assafa Tufa, Böhm Simon, Mittal Anshumali, Seeger Markus A, Bordignon Enrica (2017), Exploring conformational equilibria of a heterodimeric ABC transporter, in eLife
, 6, 1-2.
Hürlimann Lea M, Corradi Valentina, Hohl Michael, Bloemberg Guido V, Tieleman D Peter, Seeger Markus A (2016), The heterodimeric ABC transporter EfrCD mediates multidrug efflux in Enterococcus faecalis., in Antimicrobial agents and chemotherapy
, 60(9), 5400-5411.
Bukowska Magdalena A, Hohl Michael, Geertsma Eric R, Hürlimann Lea M, Grütter Markus G, Seeger Markus A (2015), A Transporter Motor Taken Apart: Flexibility in the Nucleotide Binding Domains of a Heterodimeric ABC Exporter., in Biochemistry
, 54(19), 3086-99.
Marino Jacopo, Hohl Michael, Seeger Markus A, Zerbe Oliver, Geertsma Eric R (2015), Bicistronic mRNAs to enhance membrane protein overexpression., in Journal of molecular biology
, 427(4), 943-54.
Eicher Thomas, Seeger Markus A, Anselmi Claudio, Zhou Wenchang, Brandstätter Lorenz, Verrey François, Diederichs Kay, Faraldo-Gómez José D, Pos Klaas M (2014), Coupling of remote alternating-access transport mechanisms for protons and substrates in the multidrug efflux pump AcrB., in eLife
, 3, 0.
Hohl Michael, Hürlimann Lea M, Böhm Simon, Schöppe Jendrik, Grütter Markus G, Bordignon Enrica, Seeger Markus A (2014), Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter., in Proceedings of the National Academy of Sciences of the United States of America
, 111(30), 11025-30.
Membrane transporters play a central role in every living cell. ATP-binding cassette (ABC) transporters for example are found in all phyla of life and use the energy from binding and hydrolysis of ATP to shuttle a wide variety of substrates across the membrane. Many ABC exporters actively extrude drugs and thereby contribute to the development of multidrug resistance in cancers and pathogenic microorganisms which causes severe problems in the treatment of bacterial infections and chemotherapy. Recently, we solved the crystal structure of the heterodimeric ABC exporter TM287/288 from Thermotoga maritima at a resolution of 2.9 Å. Biochemical analyses revealed that TM287/288 exhibits ATPase activity both in detergent-solution and reconstituted in membranes and is capable of extruding a variety of drugs including the anticancer agent daunomycin. Currently, TM287/288 and close bacterial homologues serve us as ideal model systems to investigate the multidrug transport mechanism and substrate recognition of ABC exporters at a molecular level. Crystal structures provide highly accurate snapshots of proteins under study, but are themselves not sufficient to understand the transport mechanism. To overcome this limitation, we use electron paramagnetic resonance (EPR) spectroscopy and single molecule FRET to study the conformational cycling of the membrane transporters both in detergent solution as well as in lipid membranes. Engineered disulfide cross-links and targeted mutations are introduced to trap ABC transporters in defined conformational states. By combining X-ray crystallography and biochemical methods, we aim to establish structure-function relationships which will ultimately allow for the detailed description of the transport cycle of heterodimeric ABC transporters. Importantly, half of the approximately 40 human ABC transporters are heterodimers like TM287/288. Therefore, our studies also contribute to the molecular understanding of mammalian ABC transporters of high clinical relevance.Tuberculosis is one of the world’s most pressing health problems, and the spread of multidrug resistant Mycobacterium tuberculosis strains makes it increasingly difficult to treat infections with the available arsenal of antibiotics. Drug resistance mediated by efflux pumps of M. tuberculosis has received increasing attention in the past decade and a few transport systems have been examined, mainly using microbiological methods. However, biochemical and structural information about these drug efflux pumps is lacking. Building on methods that I have established to study the structure and function of TM287/288, I aim to purify and characterize membrane transporters of M. tuberculosis that are involved in drug efflux and the export of virulence factors. The purified efflux pumps will be characterized at the biochemical and structural level using substrate binding assays and X-ray crystallography and functionally reconstituted in proteoliposomes to perform transport assays in vitro. Drug transport will also be studied in vivo by expressing the efflux proteins in Lactococcus lactis and Mycobacterium smegmatis. Targeted transporter gene deletions in M. tuberculosis will be characterized with respect to susceptibility to antitubercular drugs and in virulence assays using macrophage growth assays and a mouse model for tuberculosis. In summary, my research aims to answer the question how exactly transporters are capable of shuttling drugs and virulence factors across the membrane of pathogenic bacteria such as M. tuberculosis.