title: Cysteine-scanning mutagenesis and thiol modification of the Rickettsia prowazekii ATP/ADP translocase: evidence that transmembrane regions I and II, but not III, are structural components of the aqueous translocation channel.
abstract: The contribution of transmembrane regions I, II, and III of the Rickettsia prowazekii ATP/ADP translocase to the structure of the putative water-filled ATP translocation channel was evaluated from the accessibility of hydrophilic, thiol-reactive, methanethiosulfonate reagents to a library of 68 independent cysteine-substitution mutants heterologously expressed in Escherichia coli. The MTS reagents used were MTSES (negatively charged) and MTSET and MTSEA (both positively charged). Mutants F036C, Y042C, and R046C (TM I), K066C and P072C (TM II), and F101C, F105C, F108C, Y113C, and P114C (TM III) had no assayable transport activity, indicating that cysteine substitution at these positions may not be tolerated. All three MTS reagents inhibit the transport of ATP in mutants of TM I (L039C, S043C, S047C, I048C) and TM II (S061C, S063C, T067C, I069C, V070C, A074C). Further, these residues appear to cluster along a single face of the transmembrane domain. Preexposure of MTS-reactive mutants S047C (TM I) and T067C (TM II) to high levels of ATP resulted in protection from MTS-mediated inhibition. This indicated that both TM I and TM II make major contributions to the structure of an aqueous ATP translocation pathway. Finally, on the basis of the lack of accessibility of charged MTS reagents to the thiol groups in mutants of TM III, it appears that TM III is not exposed to the ATP translocation channel. Cysteine substitution of residues constituting a highly conserved "phenylalanine face" in TM III resulted in ablation of ATP transport activity. Further, substituting these phenylalanine residues for either isoleucine or tyrosine also resulted in much lower transport activity, indicating that some property of phenylalanine at these positions that is not shared by cysteine, isoleucine, or tyrosine is critical to translocase activity.
authors.affiliation: Laboratory of Molecular Biology, Department of Microbiology and Immunology, University of South Alabama College of Medicine, Mobile, Alabama 36688, USA.
authors.forename: Mikhail F
authors.initials: MF
authors.lastname: Alexeyev
authors.forename: Rosemary A W
authors.initials: RA
authors.lastname: Roberts
authors.forename: Robin M
authors.initials: RM
authors.lastname: Daugherty
authors.forename: Jonathon P
authors.initials: JP
authors.lastname: Audia
authors.forename: Herbert H
authors.initials: HH
authors.lastname: Winkler
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D003545:Cysteine / Q000235:genetics*; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives* / Q000494:pharmacology; D015640:Ion Channel Gating; D007532:Isoleucine / Q000737:chemistry; D008698:Mesylates / Q000378:metabolism; D000226:Mitochondrial ADP, ATP Translocases / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics; D010649:Phenylalanine / Q000737:chemistry; D012283:Rickettsia prowazekii / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D014178:Translocation, Genetic; D014443:Tyrosine / Q000737:chemistry
chemical_list: D008698:Mesylates; D013438:Sulfhydryl Compounds; C093171:methanethiosulfonate ethylammonium; D007532:Isoleucine; D014443:Tyrosine; C074502:methanethiosulfonate; D010649:Phenylalanine; D000226:Mitochondrial ADP, ATP Translocases; D005020:Ethyl Methanesulfonate; D003545:Cysteine