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

Item Type:Article
Title:Residues of a proposed gate region in type I ATP-binding cassette import systems are crucial for function as revealed by mutational analysis
Creators Name:Weidlich, D. and Wiesemann, N. and Heuveling, J. and Wardelmann, K. and Landmesser, H. and Sani, K.B. and Worth, C.L. and Preissner, R. and Schneider, E.
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.
Keywords:ABC Transporter, Type I Importer, Positively Charged Amino Acid, Periplasmic Gate, Solute Binding Protein, Thermophile
Source:Biochimica et Biophysica Acta - Biomembranes
Page Range:2164-2172
Date:September 2013
Additional Information:Erratum in: Biochim Biophys Acta 1838(5): 1448.
Official Publication:https://doi.org/10.1016/j.bbamem.2013.05.032
PubMed:View item in PubMed

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