title: Residues of a proposed gate region in type I ATP-binding cassette import systems are crucial for function as revealed by mutational analysis.
abstract: The type I ATP-binding cassette (ABC) importer for positively charged amino acids of the thermophilic bacterium Geobacillus stearothermophilus consists of the extracellular solute binding protein, ArtJ, and a homodimer each of the transmembrane subunit, ArtM, and the nucleotide-binding and -hydrolyzing subunit, ArtP. We have investigated the functional consequences of mutations affecting conserved residues from two peptide regions in ArtM, recently proposed to form a 'gate' by which access of a substrate to the translocation path is controlled (Hollenstein et al., 2007 [14]). Transporter variants were reconstituted into proteoliposomes and assayed for ArtJ/arginine-stimulated ATPase activity. Replacement of residues from region 1 (Arg-63, Pro-66) caused no or only moderate reduction in ATPase activity. In contrast, mutating residues from gate region 2 (Lys-159, Leu-163) resulted in a substantial increase in ATPase activity which, however, as demonstrated for variants ArtM(K159I) and ArtM(K159E), is not coupled to transport. Replacing homologous residues in the closely related histidine transporter of Salmonella enterica serovar Typhimurium (HisJ-QMP2) caused different phenotypes. Mutation to isoleucine of HisQ(K163) or HisM(H172), both homologous to ArtM(K159), abolished ATPase activity. The mutations most likely caused a structural change as revealed by limited proteolysis. In contrast, substantial, albeit reduced, enzymatic activity was observed with variants of HisQ(L167→G) or HisM(L176→G), both homologous to ArtM(L163). Our study provides the first experimental evidence in favor of a crucial role of residues from the proposed gate region in type I ABC importer function.
authors.affiliation: Institut für Biologie/Bakterienphysiologie, Humboldt Universität zu Berlin, Chausseestr. 117, D-10115 Berlin, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Weidlich
authors.forename: Nicole
authors.initials: N
authors.lastname: Wiesemann
authors.forename: Johanna
authors.initials: J
authors.lastname: Heuveling
authors.forename: Kristina
authors.initials: K
authors.lastname: Wardelmann
authors.forename: Heidi
authors.initials: H
authors.lastname: Landmesser
authors.forename: Katayoun Behnam
authors.initials: KB
authors.lastname: Sani
authors.forename: Catherine L
authors.initials: CL
authors.lastname: Worth
authors.forename: Robert
authors.initials: R
authors.lastname: Preissner
authors.forename: Erwin
authors.initials: E
authors.lastname: Schneider
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011510:Proteolipids / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: D018528:ATP-Binding Cassette Transporters; D000596:Amino Acids; D001426:Bacterial Proteins; D021122:Protein Subunits; D011510:Proteolipids; D011994:Recombinant Proteins; C020485:proteoliposomes
keywords: 3-(N-morpholino)-propanesulfonic acid; ABC; ABC transporter; ATP-binding cassette; DDM; His-tag; IPTG; MOPS; OG; PMSF; Periplasmic gate; Positively charged amino acid; Solute binding protein; Thermophile; Type I importer; hexahistidine tag; isopropyl-β-D-thio-galactopyranoside; n-dodecyl-β-D-maltopyranoside; octyl-ß-D-glucopyranoside; phenylmethylsulfonylfluoride