title: Relocation of active site carboxylates in major facilitator superfamily multidrug transporter LmrP reveals plasticity in proton interactions.
abstract: The expression of polyspecific membrane transporters is one important mechanism by which cells can obtain resistance to structurally different antibiotics and cytotoxic agents. These transporters reduce intracellular drug concentrations to subtoxic levels by mediating drug efflux across the cell envelope. The major facilitator superfamily multidrug transporter LmrP from Lactococcus lactis catalyses drug efflux in a membrane potential and chemical proton gradient-dependent fashion. To enable the interaction with protons and cationic substrates, LmrP contains catalytic carboxyl residues on the surface of a large interior chamber that is formed by transmembrane helices. These residues co-localise together with polar and aromatic residues, and are predicted to be present in two clusters. To investigate the functional role of the catalytic carboxylates, we generated mutant proteins catalysing membrane potential-independent dye efflux by removing one of the carboxyl residues in Cluster 1. We then relocated this carboxyl residue to six positions on the surface of the interior chamber, and tested for restoration of wildtype energetics. The reinsertion at positions towards Cluster 2 reinstated the membrane potential dependence of dye efflux. Our data uncover a remarkable plasticity in proton interactions in LmrP, which is a consequence of the flexibility in the location of key residues that are responsible for proton/multidrug antiport.
authors.affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK.
authors.forename: Asha V
authors.initials: AV
authors.lastname: Nair
authors.affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK.
authors.forename: Himansha
authors.initials: H
authors.lastname: Singh
authors.affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK.
authors.forename: Sagar
authors.initials: S
authors.lastname: Raturi
authors.affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK.
authors.forename: Arthur
authors.initials: A
authors.lastname: Neuberger
authors.affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Tong
authors.affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK.
authors.forename: Ning
authors.initials: N
authors.lastname: Ding
authors.affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK.
authors.forename: Kelvin
authors.initials: K
authors.lastname: Agboh
authors.affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK.
authors.forename: Hendrik W
authors.initials: HW
authors.lastname: van Veen
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002264:Carboxylic Acids / Q000378:metabolism*; D020134:Catalytic Domain; D002412:Cations / Q000378:metabolism; D013294:Lactococcus lactis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D017433:Protein Structure, Secondary; D011522:Protons
chemical_list: D001426:Bacterial Proteins; D002264:Carboxylic Acids; D002412:Cations; C096807:LmrP protein, Lactococcus lactis; D026901:Membrane Transport Proteins; D011522:Protons