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His₂₀ Provides the Sole Functionally Significant Side Chain in the Essential TonB Transmembrane Domain

Ray A. Larsen,¹ Gail E. Deckert,² Kyle A. Kastead,³ Surendranathan Devanathan,³ Kimberly L. Keller,^1, and Kathleen Postle^2,3*

Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio 43403,¹ School of Molecular Biosciences, Washington State University, Pullman, Washington 99164,² Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802³

Received 20 December 2006/ Accepted 23 January 2007

The cytoplasmic membrane protein TonB couples the protonmotive force of the cytoplasmic membrane to active transport across the outer membrane of Escherichia coli. The uncleaved amino-terminal signal anchor transmembrane domain (TMD; residues 12 to 32) of TonB and the integral cytoplasmic membrane proteins ExbB and ExbD are essential to this process, with important interactions occurring among the several TMDs of all three proteins. Here, we show that, of all the residues in the TonB TMD, only His₂₀ is essential for TonB activity. When alanyl residues replaced all TMD residues except Ser₁₆ and His₂₀, the resultant "all-Ala Ser₁₆ His₂₀" TMD TonB retained 90% of wild-type iron transport activity. Ser₁₆Ala in the context of a wild-type TonB TMD was fully active. In contrast, His₂₀Ala in the wild-type TMD was entirely inactive. In more mechanistically informative assays, the all-Ala Ser₁₆ His₂₀ TMD TonB unexpectedly failed to support formation of disulfide-linked dimers by TonB derivatives bearing Cys substitutions for the aromatic residues in the carboxy terminus. We hypothesize that, because ExbB/D apparently cannot efficiently down-regulate conformational changes at the TonB carboxy terminus through the all-Ala Ser₁₆ His₂₀ TMD, the TonB carboxy terminus might fold so rapidly that disulfide-linked dimers cannot be efficiently trapped. In formaldehyde cross-linking experiments, the all-Ala Ser₁₆ His₂₀ TMD also supported large numbers of apparently nonspecific contacts with unknown proteins. The all-Ala Ser₁₆ His₂₀ TMD TonB retained its dependence on ExbB/D. Together, these results suggest that a role for ExbB/D might be to control rapid and nonspecific folding that the unregulated TonB carboxy terminus otherwise undergoes. Such a model helps to reconcile the crystal/nuclear magnetic resonance structures of the TonB carboxy terminus with conformational changes and mutant phenotypes observed at the TonB carboxy terminus in vivo.

Published ahead of print on 2 February 2007.

Present address: Department of Biochemistry, University of Missouri, Columbia, MO 65211.

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