title: 3D model of the Escherichia coli multidrug transporter MdfA reveals an essential membrane-embedded positive charge.
abstract: MdfA is an Escherichia coli multidrug transporter of the major facilitator superfamily (MFS) of secondary transporters. Although several aspects of multidrug recognition by MdfA have been characterized, better understanding the detailed mechanism of its function requires structural information. Previous studies have modeled the 3D structures of MFS proteins, based on the X-ray structure of LacY and GlpT. However, because of poor sequence homology, between LacY, GlpT, and MdfA additional constraints were required for a reliable homology modeling. Using an algorithm that predicts the angular orientation of each transmembrane helix (TM) (kPROT), we obtained a remarkably similar pattern for the 12 TMs of MdfA and those of GlpT and LacY, suggesting that they all have similar helix packing. Consequently, a 3D model was constructed for MdfA by structural alignment with LacY and GlpT, using the kPROT results as an additional constraint. Further refinement and a preliminary evaluation of the model were achieved by correlated mutation analysis and the available experimental data. Surprisingly, in addition to the previously characterized membrane-embedded glutamate at position 26, the model suggests that Asp34 and Arg112 are located within the membrane, on the same face of the cavity as Glu26. Importantly, Arg112 is evolutionarily conserved in secondary drug transporters, and here we show that a positive charge at this position is absolutely essential for multidrug transport by MdfA.
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: Nadejda
authors.initials: N
authors.lastname: Sigal
authors.forename: Eyal
authors.initials: E
authors.lastname: Vardy
authors.forename: Shahar
authors.initials: S
authors.lastname: Molshanski-Mor
authors.forename: Asa
authors.initials: A
authors.lastname: Eitan
authors.forename: Yitzhak
authors.initials: Y
authors.lastname: Pilpel
authors.forename: Shimon
authors.initials: S
authors.lastname: Schuldiner
authors.forename: Eitan
authors.initials: E
authors.lastname: Bibi
mesh_terms: D000465:Algorithms; D001120:Arginine / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary
chemical_list: D029968:Escherichia coli Proteins; C118438:Mdfa protein, E coli; D026901:Membrane Transport Proteins; D001120:Arginine