His₂₀Provides the Sole Functionally Significant Side Chain in the Essential TonB Transmembrane Domain

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 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 His 20 is essential for TonB activity. When alanyl residues replaced all TMD residues except Ser 16 and His 20 , the resultant "all-Ala Ser 16 His 20 " TMD TonB retained 90% of wild-type iron transport activity. Ser 16 Ala in the context of a wild-type TonB TMD was fully active. In contrast, His 20 Ala in the wild-type TMD was entirely inactive. In more mechanistically informative assays, the all-Ala Ser 16 His 20 TMD TonB unexpectedly failed to support formation of disulfidelinked 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 Ser 16 His 20 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 Ser 16 His 20 TMD also supported large numbers of apparently nonspecific contacts with unknown proteins. The all-Ala Ser 16 His 20 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.The gram-negative envelope is characterized by two concentric membranes, the inner or cytoplasmic membrane (CM) and an outer, asymmetric lipopolysaccharide and phospholipid leaflet known as the outer membrane (OM). This OM retards the passage of various hydrophobic toxins and yet allows diffusion of hydrophilic nutrients through aqueous channels provided by porin proteins. While most of the metabolic demands of gram-negative bacteria can be met by diffusible nutrients, iron demand presents a complication. In neutral oxidizing environments, bioavailable iron is limiting. To obtain it, most microbes rely upon the synthesis and secretion of high-affinity iron chelators called siderophores to capture ferric ions. Because the resultant iron-siderophore complexes exceed the diffusion limit of the OM, efficient iron retrieval depends on transporters with subnanomolar affinities for the siderophores.Release (transport) of the tightly bound ligand into the periplasmic space requires energy. Because envelope physiology and architecture preclude energy production at the OM, these transp...

Deletion and Substitution Analysis of theEscherichia coliTonB Q160 Region

“…One hypothesis is that the (broadly defined) TonB carboxy terminus is held in an unstructured or disordered conformation by ExbB/ExbD to allow the induced-fit recognition of ligand-bound OM transporters (43,63). This hypothesis is consistent with much of our knowledge about protein disorder (14) and about TonB protein.…”

“…Consistent with these ideas, large regions of TonB are predicted to be disordered (43), and in the NMR structure of the monomeric TonB carboxy terminus (residues 101 to 239), residues from 101 to 151 are indeed disordered (59). It seems reasonable to speculate that ExbD, which has the same topology as TonB, may serve as the chaperone to manage the various conformational changes (possibly disorder-order transitions) that TonB goes through during an energy transduction cycle (47).…”

Solute Carrier 11 Cation Symport Requires Distinct Residues in Transmembrane Helices 1 and 6

“…It seems thus unlikely that this His residue could either bind Me 2ϩ directly, or simply be part of a relay or channel enabling H ϩ movement across the membrane (50,51). Instead, (de)protonation could favor a conformation facilitating Me 2ϩ translocation and transporter cycling; TonB His 20 is an example of a TM His moiety required for transport cycling and the energy transduction event (52). The SLC11-specific TMS6 His residue is part of another conserved motif (MPH) (with exceptions in plasmodia, plants, and fungi homologs) not found in the SLC11 phylogenetic outgroup (V(P/G)Y) (10), suggesting by analogy with TMS1 that the TMS6 motif (MPH) may represent another signature for metal transport.…”

TonB-dependent maltose transport by Caulobacter crescentus

“…The N-terminal fragment extending into the cytoplasm in C. crescentus TonB is longer (24 residues) than in E. coli TonB (11 residues). The hydrophobic, presumably transmembrane region contains a histidine residue which is conserved in the two C. crescentus TonB proteins; this residue (number 20) plays an essential role in E. coli TonB activity (Larsen et al, 2007, Larsen & Postle 2001Traub et al, 1993). Since this newly identified tonB gene is at a locus that was previously assigned to another gene with opposite polarity (cc2334), we refer to it here as cc2334a (tonB1) (Fig.…”