Conserved Residues Ser16 and His20 and Their Relative Positioning Are Essential for TonB Activity, Cross-linking of TonB with ExbB, and the Ability of TonB to Respond to Proton Motive Force

Ray A. Larsen, Kathleen Postle

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48 Scopus citations

Abstract

The cytoplasmic membrane protein TonB couples the proton electrochemical potential of the cytoplasmic membrane to transport events at the outer membrane of Gram-negative bacteria. The amino-terminal signal anchor of TonB and its interaction with the cytoplasmic membrane protein ExbB are essential to this process. The TonB signal anchor is predicted to form an α-helix, with a conserved face comprised of residues Ser16, His20, Leu27, and Ser31. Deletion of either Ser16 or His20 or of individual intervening but not flanking residues rendered TonB inactive and unable to assume a proton motive force-dependent conformation. In vivo formaldehyde cross-linking experiments revealed that the ability of this subset of mutants to form a characteristic heterodimer with ExbB was greatly diminished. Replacement of residues 17-19 by three consecutive alanines produced a wild type TonB allele, indicating that the intervening residues (Val, Cys, and Ile) contributed only to spacing. These data indicated that the spatial relationship of Ser16 to His20 was essential to function and suggested that the motif HXXXS defines the minimal requirement for the coupling of TonB to the cytoplasmic membrane electrochemical gradient. Deletion of Trp11 resulted in a TonB that remained active yet was unable to cross-link with ExbB. Because Trp 11 was demonstrably not involved in the actual cross-linking, these results suggest that the TonB/ExbB interaction detected by cross-linking occurred at a step in the energy transduction cycle distinct from the coupling of TonB to the electrochemical gradient.

Original languageEnglish (US)
Pages (from-to)8111-8117
Number of pages7
JournalJournal of Biological Chemistry
Volume276
Issue number11
DOIs
StatePublished - Mar 16 2001

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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