Role of the Transmembrane Domain in the Stability of TrwB, an Integral Protein Involved in Bacterial Conjugation

Hormaeche, Itsaso; Iloro, Ibón; Arrondo, José Luis R.; Goñi, Félix M.; Cruz, Fernando de la; Alkorta, Itziar

doi:10.1074/jbc.m310422200

Cited by 27 publications

(25 citation statements)

References 33 publications

(42 reference statements)

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“…This result is consistent with the failure of D⌬2-134, Di⌬7-134, and G-D to exhibit conjugative dominance (Table 2 and data not shown). The importance of the Nterminal domains is further attested by previous studies showing that the N-terminally-truncated CP TrwB⌬N70 has altered stability and nucleotide-binding ability (23,24) and that both TrwB⌬N70 and N-terminally-truncated mutants of RP4 TraG exhibit monomeric behavior in vitro (22,47). The K6 and I59 mutants reveal that the N-terminal region of TraD is also important for assembly beyond the dimer stage: these mutants bound wild-type TraD and formed homodimers by cross-linking but did not form the larger cross-linked structures observed for TraD and TraDiN702 even when the F plasmid was present.…”

Section: Discussionmentioning

confidence: 96%

In Vivo Oligomerization of the F Conjugative Coupling Protein TraD

et al. 2007

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Type IV secretory systems are a group of bacterial transporters responsible for the transport of proteins and nucleic acids directly into recipient cells. Such systems play key roles in the virulence of some pathogenic organisms and in conjugation-mediated horizontal gene transfer. Many type IV systems require conserved "coupling proteins," transmembrane polypeptides that are critical for transporting secreted substrates across the cytoplasmic membrane of the bacterium. In vitro evidence suggests that the functional form of coupling proteins is a homohexameric, ring-shaped complex. Using a library of tagged mutants, we investigated the structural and functional organization of the F plasmid conjugative coupling protein TraD by coimmunoprecipitation, cross-linking, and genetic means. We present direct evidence that coupling proteins form stable oligomeric complexes in the membranes of bacteria and that the formation of some of these complexes requires other F-encoded functions. Our data also show that different regions of TraD play distinct roles in the oligomerization process. We postulate a model for in vivo oligomerization and discuss the probable participation of individual domains of TraD in each step.

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Section: Discussionmentioning

confidence: 96%

In Vivo Oligomerization of the F Conjugative Coupling Protein TraD

et al. 2007

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“…Several T4CPs bind ssDNA and dsDNA substrates nonspecifically in vitro, and TrwB R388 also oligomerizes upon DNA binding in vitro (59,257). Finally, the TM domain of TrwB R388 contributes to hexamer formation and influences nucleotide binding properties (134,135).…”

Section: Biochemical and Structural Properties Of Paradigmatic T4cpsmentioning

confidence: 99%

Biological Diversity of Prokaryotic Type IV Secretion Systems

Martı́nez

Christie

2009

Microbiol Mol Biol Rev

621

780

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SUMMARY Type IV secretion systems (T4SS) translocate DNA and protein substrates across prokaryotic cell envelopes generally by a mechanism requiring direct contact with a target cell. Three types of T4SS have been described: (i) conjugation systems, operationally defined as machines that translocate DNA substrates intercellularly by a contact-dependent process; (ii) effector translocator systems, functioning to deliver proteins or other macromolecules to eukaryotic target cells; and (iii) DNA release/uptake systems, which translocate DNA to or from the extracellular milieu. Studies of a few paradigmatic systems, notably the conjugation systems of plasmids F, R388, RP4, and pKM101 and the Agrobacterium tumefaciens VirB/VirD4 system, have supplied important insights into the structure, function, and mechanism of action of type IV secretion machines. Information on these systems is updated, with emphasis on recent exciting structural advances. An underappreciated feature of T4SS, most notably of the conjugation subfamily, is that they are widely distributed among many species of gram-negative and -positive bacteria, wall-less bacteria, and the Archaea. Conjugation-mediated lateral gene transfer has shaped the genomes of most if not all prokaryotes over evolutionary time and also contributed in the short term to the dissemination of antibiotic resistance and other virulence traits among medically important pathogens. How have these machines adapted to function across envelopes of distantly related microorganisms? A survey of T4SS functioning in phylogenetically diverse species highlights the biological complexity of these translocation systems and identifies common mechanistic themes as well as novel adaptations for specialized purposes relating to the modulation of the donor-target cell interaction.

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“…9, which is published as supporting information on the PNAS web site). At 42°C, the rate of ATP hydrolysis was half the optimal value, and at 50°C the protein was inactive, probably because of thermal denaturation (20). Given the structural similarity between TrwB⌬N70 and hexameric helicases such as T7 DNA helicase, and the preference of the latter for dTTP instead of ATP, we checked TrwB⌬N70 NTP specificity for hydrolysis by using a colorimetric assay.…”

Section: Trwb⌬n70 Is a Dna-dependent Atpase Active Only At Acidic Ph mentioning

confidence: 99%

TrwB, the coupling protein involved in DNA transport during bacterial conjugation, is a DNA-dependent ATPase

Tato

Zunzunegui

Cruz

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

104

122

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Bacterial conjugation is an example of macromolecular trafficking between cells, based on the translocation of single-stranded DNA across membranes through a type IV secretion system. TrwB⌬N70 is the soluble domain of TrwB, an essential integral membrane protein that couples the relaxosome (a nucleoprotein complex) to the DNA transport apparatus in plasmid R388 conjugation. TrwB⌬N70 crystallographic structure revealed a hexamer with six equivalent subunits and a central channel. In this work, we characterize a DNA-dependent ATPase activity for TrwB⌬N70. The protein displays positive cooperativity for ATP hydrolysis, with at least three catalytic sites involved. The activity is sensitive to pH and salt concentration, being more active at low pH values. The effective oligonucleotide size required for activation of the ATPase function is between 40 and 45 nucleotides, and the same length is required for the formation of high-molecular-weight TrwB⌬N70 -DNA complexes, as observed by gel filtration chromatography. A mutation in a tryptophan residue (W216A), placed in the central pore formed by the hexameric structure, resulted in a protein that did not hydrolyze ATP. In addition, it exerted a dominant negative effect, both on R388 conjugation frequency and ATP hydrolysis, underscoring the multimeric state of the protein. ATP hydrolysis was not coupled to a DNA unwinding activity under the tested conditions, which included forked DNA substrates. These results, together with TrwB structural similarity to F 1-ATPase, lead us to propose a mechanism for TrwB as a DNA-translocating motor. molecular motor ͉ DNA transfer C onjugative plasmids provide a main route for acquisition of new genetic information in bacteria. Bacterial conjugation systems (including the related Vir system that Agrobacterium tumefaciens uses to transfer DNA to plants) show similarities to both DNA replication and protein transport systems (1, 2). The translocated substrate is a nucleoprotein particle that crosses the bacterial envelope into other bacterial or eukaryotic cells, crossing the kingdom boundaries. A two-step mechanism for DNA transport was proposed (2), in which conjugation is visualized as a DNA rolling-circle replication process linked to a type IV protein secretion system. Conjugation is encoded by two gene clusters in most conjugative plasmids. One cluster (dtr) encodes the DNA transfer replication proteins, and the other (mpf ) codes for the proteins that assemble the type IV protein secretion system channel. R388 is a 34-kb plasmid, which displays the simplest known dtr region, encoding only three proteins (TrwA, TrwB, and TrwC). The mpf region codes for 11 proteins (TrwD-N) involved in the formation of an extracellular pilus and a membrane-associated complex that translocates the nucleoprotein substrate across membranes.TrwB, an integral membrane and DNA-binding protein, is a key player in R388 conjugation (3-6). It has a counterpart in all conjugative systems, and it is thought to be responsible for recruiting the relaxosome DNA-pr...

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Role of the Transmembrane Domain in the Stability of TrwB, an Integral Protein Involved in Bacterial Conjugation

Cited by 27 publications

References 33 publications

In Vivo Oligomerization of the F Conjugative Coupling Protein TraD

In Vivo Oligomerization of the F Conjugative Coupling Protein TraD

Biological Diversity of Prokaryotic Type IV Secretion Systems

TrwB, the coupling protein involved in DNA transport during bacterial conjugation, is a DNA-dependent ATPase

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