The authors regret that References [25–31] are in the wrong order and that the correct reference [30] is missing. The corrected references are as follows: [25] N.J. Gleason, V.V. Vostrikov, D.V. Greathouse, R.E. Koeppe, Buried lysine, but not arginine, titrates and alters transmembrane helix tilt, Proc. Natl. Acad. Sci. (USA) 110 (2013) 1692–1695. [26] J. Cui, S. Qasim, A.L. Davidson, Uncoupling substrate transport from ATP hydrolysis in the Escherichia coli maltose transporter. J. Biol. Chem. 285 (2010) 39986–39993. [27] K.-M.Y. Covitz, C.H. Panagiotidis, L.-I. Hor, M. Reyes, N.A. Treptow, H.A. Shuman, Mutations that alter the transmembrane signalling pathway in an ATP binding cassette (ABC) transporter. EMBO-J. 13 (1994) 1752–1759. [28] Chen, S. Sharma, F.A. Quiocho, A.L. Davidson, Trapping the transition state of an ATP-binding cassette transporter: evidence for a concerted mechanism of maltose transport. Proc. Natl. Acad. Sci. (USA) 98 (2001) 1525–1530. [29] I.L. Urbatsch, B. Sankaran, J. Weber, A.E. Senior, P-glycoprotein is stably inhibited by vanadate-induced trapping of nucleotide at a single catalytic site. J. Biol. Chem. 270 (1995) 19383–19390. [30] M.L. Oldham, J. Chen, Snapshots of the maltose transporter during ATP hydrolysis. Proc. Natl. Acad. Sci. (USA) 108 (2011) 15152–15156. [31] Davidson, H.A. Shuman, H. Nikaido, Mechanism of maltose transport in Escherichia coli: transmembrane signaling by periplasmic binding proteins. Proc. Natl. Acad. Sci. USA 89 (1992) 2360–2364. [32] A.D. Gould, P.G. Telmer, B.H. Shilton, Stimulation of themaltose transporter ATPase by unliganded maltose binding protein. Biochemistry 48 (2009) 8051–8061. The authors would like to apologize for any inconvenience caused.

The conformational dynamics of the histidine ABC transporter HisQMP2 from Salmonella enterica serovar Typhimurium, reconstituted into liposomes, is studied by site-directed spin labeling and double electron–electron resonance spectroscopy in the absence of nucleotides, in the ATP-bound, and in the post-hydrolysis state. The results show that the inter-dimer distances as measured between the Q-loops of HisP2 in the intact transporter resemble those determined for the maltose transporter in all three states of the hydrolysis cycle. Only in the presence of liganded HisJ the closed conformation of the nucleotide binding sites is achieved revealing the transmembrane communication of the presence of substrate. Two conformational states can be distinguished for the periplasmic moiety of HisQMP2 as detected by differences in distributions of interspin distances between positions 86 and 96 or 104 and 197. The observed conformational changes are correlated to proposed open, semi-open and closed conformations of the nucleotide binding domains HisP2. Our results are in line with a rearrangement of transmembrane helices 4 and 4′ of HisQM during the closed to the semi-open transition of HisP2 driven by the reorientation of the coupled helices 3a and 3b to occur upon hydrolysis.

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.

ATP-binding cassette (ABC) transport systems mediate the translocation of solutes across biological membranes at the expense of ATP. They share a common modular architecture comprising two pore-forming transmembrane domains and two nucleotide binding domains. In prokaryotes, ABC transporters are involved in the uptake of a large variety of chemicals, including nutrients, osmoprotectants and signal molecules. In pathogenic bacteria, some ABC importers are virulence factors. Canonical ABC import systems require an additional component, a substrate-specific receptor or binding protein for function. Interaction of the liganded receptor with extracytoplasmic loop regions of the transmembrane domains initiate the transport cycle. In this review we summarize the current knowledge on receptor–transporter interplay provided by crystal structures as well as by biochemical and biophysical means. In particular, we focus on the maltose/maltodextrin transporter of enterobacteria and the transporters for positively charged amino acids from the thermophile Geobacillus stearothermophilus and Salmonella enterica serovar Typhimurium.

ABSTRACT In the thermophile Geobacillus stearothermophilus , the uptake of basic amino acids is mediated by an ABC transporter composed of the substrate binding protein (receptor) ArtJ and a homodimer each of the pore-forming subunit, ArtM, and the nucleotide-binding subunit, ArtP. We recently identified two putative binding sites in ArtJ that might interact with the Art(MP) 2 complex, thereby initiating the transport cycle (A. Vahedi-Faridi et al., J. Mol. Biol. 375:448-459, 2008). Here we investigated the contribution of charged amino acid residues in the second extracellular loop of ArtM to contact with ArtJ. Our results demonstrate a crucial role for residues K177, R185, and E188, since mutations to oppositely charged amino acids or glutamine led to a complete loss of ArtJ-stimulated ATPase activity of the complex variants in proteoliposomes. The defects could not be suppressed by ArtJ variants carrying mutations in site I (K39E and K152E) or II (E163K and D170K), suggesting a more complex interplay than that by a single salt bridge. These findings were supported by cross-linking assays demonstrating physical proximity between ArtJ(N166C) and ArtM(E182C). The importance of positively charged residues for receptor-transporter interaction was underscored by mutational analysis of the closely related transporter HisJ/LAO-HisQMP 2 of Salmonella enterica serovar Typhimurium. While transporter variants with mutated positively charged residues in HisQ displayed residual ATPase activities, corresponding mutants of HisM could no longer be stimulated by HisJ/LAO. Interestingly, the ATPase activity of the HisQM(K187E)P 2 variant was inhibited by l - and d -histidine in detergent, suggesting a role of the residue in preventing free histidine from gaining access to the substrate binding site within HisQM.