title: Taking the Escherichia coli TonB transmembrane domain "offline"? Nonprotonatable Asn substitutes fully for TonB His20.
abstract: The TonB system of Gram-negative bacteria uses the proton motive force (PMF) of the cytoplasmic membrane to energize active transport of nutrients across the outer membrane. The single transmembrane domain (TMD) anchor of TonB, the energy transducer, is essential. Within that TMD, His20 is the only TMD residue that is unable to withstand alanine replacement without a loss of activity. H20 is required for a PMF-dependent conformational change, suggesting that the importance of H20 lies in its ability to be reversibly protonated and deprotonated. Here all possible residues were substituted at position 20 (H20X substitutions). The His residue was also relocated throughout the TonB TMD. Surprisingly, Asn, a structurally similar but nonprotonatable residue, supported full activity at position 20; H20S was very weakly active. All the remaining substitutions, including H20K, H20R, H20E, and H20D, the obvious candidates to mimic a protonated state or support proton translocation, were inactive. A second-site suppressor, ExbB(A39E), indiscriminately reactivated the majority of H20 substitutions and relocations, including H20V, which cannot be made protonatable. These results suggested that the TonB TMD was not on a proton conductance pathway and thus only indirectly responds to PMF, probably via ExbD.
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Cheryl
authors.initials: C
authors.lastname: Swayne
authors.forename: Kathleen
authors.initials: K
authors.lastname: Postle
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001216:Asparagine / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary
chemical_list: D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C489181:tonB protein, E coli; D006639:Histidine; D001216:Asparagine