His₂₀ provides the sole functionally significant side chain in the essential TonB transmembrane domain

Department of Biological Sciences, Bowling Green State University, Bowling Green, OH 42402; School of Molecular Biosciences, Washington State University, Pullman, WA 99164

^* To whom correspondence should be addressed. Email: postle{at}psu.edu.

	Abstract

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 12-32) of TonB and the integral cytoplasmic membrane proteins ExbB and ExbD are essential to this process, with important interactions occurring among the several transmembrane domains 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 wildtype iron transport activity. Ser₁₆Ala in the context of a wildtype TonB TMD was fully active. In contrast, His₂₀Ala in the wildtype 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 of 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 crosslinking experiments, the all-Ala Ser₁₆, His₂₀ TMD also supported large numbers of apparently non-specific contacts with unknown proteins. The all-Ala Ser₁₆, His₂₀TMD TonB retained its dependence on ExbB/D. Together, these results suggest that a role of ExbB/D might be to control rapid and non-specific folding that the unregulated TonB carboxy terminus otherwise undergoes. Such a model helps to reconcile the crystal/NMR structures of the TonB carboxy terminus with conformational changes and mutant phenotypes observed at the TonB carboxy terminus in vivo.