Residues of a proposed gate region in type I ATP-binding cassette import systems are crucial for function as revealed by mutational analysis

• Published in: Biochimica et biophysica acta 2013-09, Vol.1828 (9), p.2164-2172
• Main Authors: Weidlich, Daniela, Wiesemann, Nicole, Heuveling, Johanna, Wardelmann, Kristina, Landmesser, Heidi, Sani, Katayoun Behnam, Worth, Catherine L., Preissner, Robert, Schneider, Erwin
• Format: Article
• Language: English
• Subjects: ABC transporter; Amino Acid Sequence; Amino Acid Substitution; Amino Acids - chemistry; Amino Acids - genetics; ATP-Binding Cassette Transporters - chemistry; ATP-Binding Cassette Transporters - genetics; ATP-Binding Cassette Transporters - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Geobacillus stearothermophilus - chemistry; Geobacillus stearothermophilus - genetics; Geobacillus stearothermophilus - metabolism; Models, Molecular; Molecular Sequence Data; Mutation; Periplasmic gate; Positively charged amino acid; Protein Subunits - chemistry; Protein Subunits - genetics; Protein Subunits - metabolism; Proteolipids - chemistry; Proteolipids - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Salmonella typhimurium - chemistry; Salmonella typhimurium - genetics; Sequence Homology, Amino Acid; Solute binding protein; Structure-Activity Relationship; Thermophile; Type I importer
• ISSN: 0005-2736; 0006-3002; 1879-2642
• DOI: 10.1016/j.bbamem.2013.05.032

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. [Display omitted] •Role of gate regions in type I ABC transporter Art(MP)2 is studied by mutagenesis.•Mutations of residues from gate region 2 cause increase in ATPase activity.•Increase in ATPase activity is uncoupled from transport.•Mutations in the homologous HisQMP2 transporter cause different phenotypes.•Our study supports role of gate region in transporter function.