Mapping the Interactions between Escherichia coli TolQ Transmembrane Segments

Zhang, Xiang Y.-Z.; Goemaere, Emilie L.; Seddiki, Nabila; Celia, Hervé; Gavioli, Marthe; Cascales, Éric; Lloubès, Roland

doi:10.1074/jbc.m110.192773

Cited by 22 publications

(29 citation statements)

References 48 publications

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“…However, the cellular ExbB-ExbD-TonB ratios seen in vivo are at odds with an abundance of in vitro data, suggesting that the basic composition of complexes of ExbB and ExbD or its paralogues consist of 4 ExbB and 2 ExbD, giving a ratio of 2:1, not 7:2 (35,52,53). Consistent with the 4:2 composition of an in vitro ExbB-ExbD complex, ExbB formaldehyde cross-links in vivo as a dimer of homodimers to yield ExbB 4 (24), and the carboxy terminus of ExbD cross-links as a homodimer, ExbD 2 (32).…”

Section: Discussionmentioning

confidence: 79%

From Homodimer to Heterodimer and Back: Elucidating the TonB Energy Transduction Cycle

2015

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The TonB system actively transports large, scarce, and important nutrients through outer membrane (OM) transporters of Gram-negative bacteria using the proton gradient of the cytoplasmic membrane (CM). In Escherichia coli, the CM proteins ExbB and ExbD harness and transfer proton motive force energy to the CM protein TonB, which spans the periplasmic space and cyclically binds OM transporters. TonB has two activity domains: the amino-terminal transmembrane domain with residue H20 and the periplasmic carboxy terminus, through which it binds to OM transporters. TonB is inactivated by all substitutions at residue H20 except H20N. Here, we show that while TonB trapped as a homodimer through its amino-terminal domain retained full activity, trapping TonB through its carboxy terminus inactivated it by preventing conformational changes needed for interaction with OM transporters. Surprisingly, inactive TonB H20A had little effect on homodimerization through the amino terminus and instead decreased TonB carboxy-terminal homodimer formation prior to reinitiation of an energy transduction cycle. That result suggested that the TonB carboxy terminus ultimately interacts with OM transporters as a monomer. Our findings also suggested the existence of a separate equimolar pool of ExbD homodimers that are not in contact with TonB. A model is proposed where interaction of TonB homodimers with ExbD homodimers initiates the energy transduction cycle, and, ultimately, the ExbD carboxy terminus modulates interactions of a monomeric TonB carboxy terminus with OM transporters. After TonB exchanges its interaction with ExbD for interaction with a transporter, ExbD homodimers undergo a separate cycle needed to re-energize them. IMPORTANCECanonical mechanisms of active transport across cytoplasmic membranes employ ion gradients or hydrolysis of ATP for energy. Gram-negative bacterial outer membranes lack these resources. The TonB system embodies a novel means of active transport across the outer membrane for nutrients that are too large, too scarce, or too important for diffusion-limited transport. A proton gradient across the cytoplasmic membrane is converted by a multiprotein complex into mechanical energy that drives high-affinity active transport across the outer membrane. This system is also of interest since one of its uses in pathogenic bacteria is for competition with the host for the essential element iron. Understanding the mechanism of the TonB system will allow design of antibiotics targeting iron acquisition.T he ability to acquire iron is the basis of a tug-of-war between host and bacterial pathogen (1, 2). Successful pathogens can acquire iron from their hosts, and in the case of Gram-negative bacteria, that is usually mediated by the TonB system, thus making the understanding of its mechanism an imperative goal (3, 4) and an attractive target for antibiotic development (5).In broad terms, the TonB system harnesses the proton motive force (PMF) of the cytoplasmic membrane (CM) to energize active transport across a ...

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Section: Discussionmentioning

confidence: 79%

From Homodimer to Heterodimer and Back: Elucidating the TonB Energy Transduction Cycle

2015

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“…It is notable that the suppressors all exchange the negatively charged Asp or Glu for the native Ala or Val residues (39,40). Similar suppressors exist between TonB/ExbB paralogues TolA/TolQ, where a TolQ TMD1 face corresponding to ExbB S34 supports similar conserved functional interactions (70,71).…”

Section: Fig 9 Summary Of Residue Substitutions In Exbb Transmembranementioning

confidence: 79%

Mutations in Escherichia coli ExbB Transmembrane Domains Identify Scaffolding and Signal Transduction Functions and Exclude Participation in a Proton Pathway

Baker

Postle

2013

J Bacteriol

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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. In Gram-negative bacteria, the TonB system couples cytoplasmic membrane (CM) proton motive force (pmf) to active transport of ligands across the outer membrane (OM) through TonB-gated transporters (TGTs) (for reviews, see references 1 to 5). The TonB system is composed of three CM proteins, TonB, ExbB, and ExbD, and several different TGTs in the OM that recognize diverse ligands. In Escherichia coli K-12, the TonB system transports ironsiderophore compounds and vitamin B 12 , while in other Gramnegative bacteria, additional nutrients, including nickel, sucrose, heme, and maltodextrin, are transported (6-9).TonB and ExbD have similar topologies of one N-terminal transmembrane domain (TMD) and a large periplasmic domain (10, 11). In contrast, ExbB contains three TMDs with a large soluble cytoplasmic loop between TMDs 1 and 2 and a soluble cytoplasmic C-terminal tail domain (12, 13). ExbB appears to be the scaffold upon which TonB and ExbD assemble, since TonB and ExbD are proteolytically unstable in the absence of ExbB, while ExbB stability is independent of TonB and ExbD (13-16; unpublished results).TonB physically interacts with OM TGTs, connecting CM pmf energy to OM ligand transport (17)(18)(19)(20). A proton pathway through ExbB, ExbD, and/or TonB TMD residues has been proposed based on the similar sequence hom...

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“…The MotAB, TolQR and ExbBD proton channels are able to transduce energy stored in the proton motive force (PMF) to allow for flagellar rotation, outer membrane (OM) biogenesis and transport processes across the OM respectively (Lloubès et al ., ; Minamino et al ., ; Noinaj et al ., ). In all these complexes the proton channel consists of three alpha helical transmembrane (TM) domains where two α‐helices are donated from the larger subunit (MotA, TolQ, ExbB) and the third α‐helix stems from the corresponding smaller subunit (MotB, TolR, ExbD) (Zhai et al ., ; Braun et al ., ; Zhang et al ., ). Although the functions of these complexes are quite diverse, they show remarkable sequence homologies within these TM domains (Cascales et al ., ; Goemaere et al ., ).…”

Section: Resultsmentioning

confidence: 97%

SiiA and SiiB are novel type I secretion system subunits controlling SPI4-mediated adhesion ofSalmonella enterica

Wille¹,

Wagner

Mittelstädt

et al. 2013

Cell Microbiol

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SummaryThe giant non-fimbrial adhesin SiiE is essential to establish intimate contact between Salmonella enterica and the apical surface of polarized epithelial cells. SiiE is secreted by a type I secretion system (T1SS) encoded by Salmonella Pathogenicity Island 4 (SPI4). We identified SiiA and SiiB as two regulatory proteins encoded by SPI4. Mutant strains in siiA or siiB still secrete SiiE, but are highly reduced in adhesion to, and invasion of polarized cells. SiiA and SiiB are inner membrane proteins with one and three transmembrane (TM) helices respectively. TM2 and TM3 of SiiB are similar to members of the ExbB/TolQ family, while the TM of SiiA is similar to MotB and a conserved aspartate residue in this TM is essential for SPI4-encoded T1SS function. Co-immunoprecipitation, bacterial two-hybrid and FRET demonstrate homoand heterotypic protein interactions for SiiA and SiiB. SiiB, but not SiiA also interacts with the SPI4-T1SS ATPase SiiF. The integrity of the Walker A box in SiiF was required for SiiB-SiiF interaction and SiiF dimer formation. Based on these data, we describe SiiA and SiiB as new, exclusively virulence-associated members of the Mot/Exb/Tol family of membrane proteins. Both proteins are involved in a novel mechanism of controlling SPI4-T1SS-dependent adhesion, most likely by formation of a proton-conducting channel.

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Mapping the Interactions between Escherichia coli TolQ Transmembrane Segments

Abstract: The tolQRAB-pal operon is conserved in Gram

Cited by 22 publications

References 48 publications

From Homodimer to Heterodimer and Back: Elucidating the TonB Energy Transduction Cycle

From Homodimer to Heterodimer and Back: Elucidating the TonB Energy Transduction Cycle

Mutations in Escherichia coli ExbB Transmembrane Domains Identify Scaffolding and Signal Transduction Functions and Exclude Participation in a Proton Pathway

SiiA and SiiB are novel type I secretion system subunits controlling SPI4-mediated adhesion ofSalmonella enterica

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