title: Effect of the F610A mutation on substrate extrusion in the AcrB transporter: explanation and rationale by molecular dynamics simulations.
abstract: The tripartite efflux pump AcrAB-TolC is responsible for the intrinsic and acquired multidrug resistance in Escherichia coli. Its active part, the homotrimeric transporter AcrB, is in charge of the selective binding of substrates and energy transduction. The mutation F610A has been shown to significantly reduce the minimum inhibitory concentration of doxorubicin and many other substrates, although F610 does not appear to interact strongly with them. Biochemical study of transport kinetics in AcrB is not yet possible, except for some β-lactams, and other techniques should supply this important information. Therefore, in this work, we assess the impact of the F610A mutation on the functionality of AcrB by means of computational techniques, using doxorubicin as substrate. We found that the compound slides deeply inside the binding pocket after mutation, increasing the strength of the interaction. During subsequent conformational alterations of the transporter, doxorubicin was either not extruded from the binding site or displaced along a direction other than the one associated with extrusion. Our study indicates how subtle interactions determine the functionality of multidrug transporters, since decreased transport might not be simplistically correlated to decreased substrate binding affinity.
authors.affiliation: CNR-IOM, Unità SLACS, S.P. Monserrato-Sestu Km 0.700, I-09042 Monserrato (CA), Italy. vargiu@dsf.unica.it
authors.forename: Attilio V
authors.initials: AV
authors.lastname: Vargiu
authors.forename: Francesca
authors.initials: F
authors.lastname: Collu
authors.forename: Robert
authors.initials: R
authors.lastname: Schulz
authors.forename: Klaas M
authors.initials: KM
authors.lastname: Pos
authors.forename: Martin
authors.initials: M
authors.lastname: Zacharias
authors.forename: Ulrich
authors.initials: U
authors.lastname: Kleinekathöfer
authors.forename: Paolo
authors.initials: P
authors.lastname: Ruggerone
mesh_terms: D019943:Amino Acid Substitution; D019295:Computational Biology; D004317:Doxorubicin / Q000378:metabolism; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011487:Protein Conformation
chemical_list: D004317:Doxorubicin; D029968:Escherichia coli Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D050505:Mutant Proteins; C083496:AcrB protein, E coli