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.

Fig 1

Revision of ExbB TMD boundaries. ExbB topology within the cytoplasmic membrane (parallel black lines) and the TOPCONS-predicted boundaries for TMDs 1, 2, and 3 are shown. The numbers in parentheses in the figure are previous ExbB TMD predictions (12, 13, 21). The majority of ExbB is localized in the cytoplasm (Cyto), as a large cytoplasmic loop and C-terminal tail. The N terminus of ExbB is in the periplasm (Peri). The general locations of the previously identified ExbB E176A TMD mutant and T148A/T181A double mutant are shown (boxes) (28).

Fig 2

[⁵⁵Fe]ferrichrome transport supported by ExbB TMD substitutions. ExbB TMD substituted mutants were expressed at chromosomal levels in strain RA1017 (ΔexbBD ΔtolQRA), and the initial rates of [⁵⁵Fe]ferrichrome transport were measured as described in Materials and Methods. Triplicate samples were taken at all time points, and the initial transport rates were calculated by linear regression. Linear regression slopes were normalized to plasmid-encoded wild-type ExbBD (pExbBD) (100%). Normalized percent activities of at least two independent experiments were averaged, and standard deviations are indicated by the error bars. ExbB TMD substitutions are presented as follows: (A) substitution of hydroxyl residues; (B) substitution of proline, charged, and the remaining polar residues; (C) substitution of glycine residues.

Fig 3

ExbB is required for conformational response of TonB to pmf and efficient TonB-ExbD assembly. Spheroplasts (Sph) or whole cells (WC) were treated with dimethyl sulfoxide (DMSO) (sph) or CCCP and subsequently not treated with proteinase K (−) or treated (+) with proteinase K (+PK) for a time course of 2, 5, 10, and 15 min, followed by the addition of the protease inhibitor (PMSF) and immediate precipitation with trichloroacetic acid (TCA). TCA-precipitated samples were resolved on 13% SDS-polyacrylamide gels and immunoblotted with anti-TonB (α TonB) antibodies. The positions of molecular mass markers (in kilodaltons) are indicated to the left of the blots. Plasmid-expressed wild-type and mutant proteins or the deletion strain are indicated to the right of each blot. The position of the TonB proteinase K-resistant ∼23-kDa fragment is indicated by an asterisk to the right of the blots. (A) ExbB facilitates initial TonB-ExbD assembly. Spheroplasts were generated from the ΔexbBD ΔtolQRA strain (RA1017), expressing pExbBD (pKP660) or pExbD (pKP1194) at chromosomal levels. RA1017 containing the empty vector, pBAD24, is shown in the bottom blot. (B) ExbB TMD mutations affect TonB conformational response to pmf. Spheroplasts were generated in RA1017 expressing plasmid-encoded ExbB TMD mutants at chromosomal levels as well as ExbD. This figure is a composite of representative immunoblots.

Fig 4

ExbB S34A and W38A TMD mutants are not dominant. ExbB mutants were coexpressed with ExbD using 0.01% l-arabinose in wild-type (WT) strain W3110. All mutant proteins were overexpressed >100-fold compared to chromosomal levels, based on serial dilutions and immunoblot analysis (data not shown). Initial rates of [⁵⁵Fe]ferrichrome transport were measured from triplicate samples and normalized to the results for strain W3110 (100%). Averaged percentages of at least two independent experiments are shown.

Fig 5

ExbB S34A and W38A reduce the TonB-ExbB formaldehyde cross-linked complex. ExbB TMD1 mutants were expressed at chromosomal levels in strain RA1017 (ΔexbBD ΔtolQRA), and parent strain W3110 (WT) served as the wild-type chromosomal control. The strain or mutations are indicated above the lanes. Cultures grown to mid-exponential phase were cross-linked with monomeric formaldehyde and solubilized in gel sample buffer at 60°C. Samples were resolved on 11% SDS-polyacrylamide gels and immunoblotted with anti-TonB antibody. The positions and compositions of complexes are indicated to the right of the blot. The positions of molecular mass standards (in kilodaltons) are indicated to the left of the blot. A shorter exposure of the same immunoblot is shown in the bottom blot for comparison of monomer levels.

Fig 6

ExbB P190A reduces ExbB tetramer + X complex formation. ExbB TMD3 mutants were expressed at chromosomal levels in strain RA1017 (ΔexbBD ΔtolQRA), and parent strain W3110 (WT) served as the wild-type chromosomal control. The strain or mutations are indicated above the lanes. Cultures grown to mid-exponential phase were cross-linked with monomeric formaldehyde and solubilized in gel sample buffer at 60°C. Samples were resolved on 13% SDS-polyacrylamide gels and immunoblotted with anti-ExbB antibody. The positions and compositions of complexes are indicated to the right of the blot. The positions of molecular mass standards are indicated to the left of the blot. A shorter exposure of the same immunoblot is shown in the bottom blot for comparison of monomer levels.

Fig 7

Conservative S34 substitutions restore low but detectable levels of the TonB-ExbB formaldehyde cross-linked complex. ExbB S34 substitutions were expressed at chromosomal levels in strain RA1017 (ΔexbBD ΔtolQRA), and parent strain W3110 (WT) served as the wild-type chromosomal control. The strain or mutations are indicated above the lanes. Cultures grown to mid-exponential phase were cross-linked with monomeric formaldehyde and solubilized in gel sample buffer at 60°C. Samples were resolved on 11% SDS-polyacrylamide gels and immunoblotted with anti-TonB antibody. The positions and compositions of complexes are indicated to the right of the blot. The positions of molecular mass standards are indicated to the left of the blot. A shorter exposure of the same immunoblot is shown in the bottom blot for comparison of TonB monomer levels. For ExbB monomer levels, the same samples were resolved on a 13% SDS-polyacrylamide gel and immunoblotted with anti-ExbB antibody.

Fig 8

Sequence alignment of ExbB, TolQ, MotA, and PomA. ExbB, TolQ, MotA, and PomA sequences were aligned using Clustal omega multiple-sequence alignment program (http://www.ebi.ac.uk/Tools/msa/clustalo/). Charged residues are shown on a gray background. Predicted ExbB TMDs from Fig. S1 in the supplemental material are indicated by a black bar above the sequence. The dashed bar indicates the predicted location of the MotA/PomA TMD1 (64). ExbB (NCBI accession no. YP_491200.1), TolQ (YP_489017.1), and MotA (YP_490152.1) sequences are from Escherichia coli K-12 strain W3110. The PomA sequence is from Vibrio alginolyticus (GenBank accession no. BAA20284.1).The importance of glycines in alignment of transmembrane domains among these proteins has been recognized previously (59, 92). Gaps introduced to maximize alignment are indicated by dashes in the sequences. Asterisks, colons, and periods indicate identical, conserved, and semiconserved residues, respectively.

Fig 9

Summary of residue substitutions in ExbB transmembrane domains. Predicted ExbB TMDs are depicted as shaded sequences. Gray residues within each sequence indicate the positions of Ala substitutions. All substituted residues are shown in gray: light gray residues were functional, and dark gray, underlined residues were nonfunctional when substituted to alanine. The corresponding amino acid numbers are listed above the residues. ExbB Y195A and N196A substitutions were previously reported (43).