title: On the mechanism of substrate specificity by resistance nodulation division (RND)-type multidrug resistance pumps: the large periplasmic loops of MexD from Pseudomonas aeruginosa are involved in substrate recognition.
abstract: Tripartite efflux systems of Gram-negative bacteria that contain an inner membrane transporter belonging to the resistance nodulation division (RND) superfamily can extrude a large variety of structurally diverse compounds. To gain an insight into the molecular mechanisms of substrate recognition by these multidrug resistance (MDR) transporters, we isolated spontaneous mutations that altered the substrate specificity of the MexCD-OprJ pump from Pseudomonas aeruginosa. These mutations enabled the pump to extrude the normally non-transported beta-lactam antibiotic carbenicillin. All amino acid substitutions were mapped to the large periplasmic loops (LPLs) of the RND proper, MexD. Q34K, E89K, A292V and P328L were found in the first LPL, located between transmembrane domains (TMD) 1 and 2, whereas F608S and N673K were contained in the second LPL, located between TMD7 and TMD8. These mutations also had a substantial impact on the MexCD-OprJ-mediated transport of numerous other substrates. Subsequent replacement of amino acid residues identified above by cysteines rendered MexCD-OprJ susceptible to inhibition by a thiol-reactive agent, MIANS. Interestingly, MIANS inhibited the transport of some (pyronin, EtBr) but not other (ANS, Leu-Nap) substrates of the pump. Our results suggest that the precise structure of the periplasmic loops of MexD determines the rate of transport of individual substrates. These results are consistent with the hypothesis that, in the case of RND transporters, the LPLs are directly implicated in substrate recognition and contain multiple sites of interaction for various structurally diverse compounds.
authors.affiliation: Essential Therapeutics Inc., 850 Maude Ave., Mountain View, CA 94043, USA.
authors.forename: Weimin
authors.initials: W
authors.lastname: Mao
authors.forename: Mark S
authors.initials: MS
authors.lastname: Warren
authors.forename: Deborah S
authors.initials: DS
authors.lastname: Black
authors.forename: Takahumi
authors.initials: T
authors.lastname: Satou
authors.forename: Takeshi
authors.initials: T
authors.lastname: Murata
authors.forename: Takeshi
authors.initials: T
authors.lastname: Nishino
authors.forename: Naomasa
authors.initials: N
authors.lastname: Gotoh
authors.forename: Olga
authors.initials: O
authors.lastname: Lomovskaya
mesh_terms: D019943:Amino Acid Substitution; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002228:Carbenicillin / Q000378:metabolism / Q000494:pharmacology; D024901:Drug Resistance, Multiple, Bacterial; D007769:Lactams / Q000378:metabolism / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D019897:Periplasm; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001425:Bacterial Outer Membrane Proteins; D007769:Lactams; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C470189:MexC protein, Pseudomonas aeruginosa; C121980:MexD protein, Pseudomonas aeruginosa; D002228:Carbenicillin