title: Mutations in Escherichia coli ExbB transmembrane domains identify scaffolding and signal transduction functions and exclude participation in a proton pathway.
abstract: The TonB system couples cytoplasmic membrane proton motive force (pmf) to active transport of diverse nutrients across the outer membrane. Current data suggest that cytoplasmic membrane proteins ExbB and ExbD harness pmf energy. Transmembrane domain (TMD) interactions between TonB and ExbD allow the ExbD C terminus to modulate conformational rearrangements of the periplasmic TonB C terminus in vivo. These conformational changes somehow allow energization of high-affinity TonB-gated transporters by direct interaction with TonB. While ExbB is essential for energy transduction, its role is not well understood. ExbB has N-terminus-out, C-terminus-in topology with three TMDs. TMDs 1 and 2 are punctuated by a cytoplasmic loop, with the C-terminal tail also occupying the cytoplasm. We tested the hypothesis that ExbB TMD residues play roles in proton translocation. Reassessment of TMD boundaries based on hydrophobic character and residue conservation among distantly related ExbB proteins brought earlier widely divergent predictions into congruence. All TMD residues with potentially function-specific side chains (Lys, Cys, Ser, Thr, Tyr, Glu, and Asn) and residues with probable structure-specific side chains (Trp, Gly, and Pro) were substituted with Ala and evaluated in multiple assays. While all three TMDs were essential, they had different roles: TMD1 was a region through which ExbB interacted with the TonB TMD. TMD2 and TMD3, the most conserved among the ExbB/TolQ/MotA/PomA family, played roles in signal transduction between cytoplasm and periplasm and the transition from ExbB homodimers to homotetramers. Consideration of combined data excludes ExbB TMD residues from direct participation in a proton pathway.
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA.
authors.forename: Kristin R
authors.initials: KR
authors.lastname: Baker
authors.forename: Kathleen
authors.initials: K
authors.lastname: Postle
mesh_terms: D019943:Amino Acid Substitution; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017494:Proton Pumps; D015398:Signal Transduction
chemical_list: D029968:Escherichia coli Proteins; C052874:ExbB protein, E coli; D050505:Mutant Proteins; D017494:Proton Pumps