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The FtsK-like motor TraB is a DNA-dependent ATPase that forms higher-order assemblies.

ABSTRACT: TraB is an FtsK-like DNA translocase responsible for conjugative plasmid transfer in mycelial Streptomyces Unlike other conjugative systems, which depend on a type IV secretion system, Streptomyces requires only TraB protein to transfer the plasmid as dsDNA. The ?-domain of this protein specifically binds to repeated 8-bp motifs on the plasmid sequence, following a mechanism that is reminiscent of the FtsK/SpoIIIE chromosome segregation system. In this work, we purified and characterized the enzymatic activity of TraB, revealing that it is a DNA-dependent ATPase that is highly stimulated by dsDNA substrates. Interestingly, we found that unlike the SpoIIIE protein, the ?-domain of TraB does not confer sequence-specific ATPase stimulation. We also found that TraB binds G-quadruplex DNA structures with higher affinity than TraB-recognition sequences (TRSs). An EM-based structural analysis revealed that TraB tends to assemble as large complexes comprising four TraB hexamers, which might be a prerequisite for DNA translocation across cell membranes. In summary, our findings shed light on the molecular mechanism used by the DNA-translocating motor TraB, which may be shared by other membrane-associated machineries involved in DNA binding and translocation.

SUBMITTER: Amado E

PROVIDER: S-EPMC6442058 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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The FtsK-like motor TraB is a DNA-dependent ATPase that forms higher-order assemblies.

Amado Eric E Muth Günther G Arechaga Ignacio I Cabezón Elena E

The Journal of biological chemistry 20190205 13

TraB is an FtsK-like DNA translocase responsible for conjugative plasmid transfer in mycelial <i>Streptomyces</i> Unlike other conjugative systems, which depend on a type IV secretion system, <i>Streptomyces</i> requires only TraB protein to transfer the plasmid as dsDNA. The γ-domain of this protein specifically binds to repeated 8-bp motifs on the plasmid sequence, following a mechanism that is reminiscent of the FtsK/SpoIIIE chromosome segregation system. In this work, we purified and charact ...[more]

PMID: 30723158

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Project description:BackgroundThe recent outbreak of severe infections with Shiga toxin (Stx) producing Escherichia coli (STEC) serotype O104:H4 highlights the need to understand horizontal gene transfer among E. coli strains, identify novel virulence factors and elucidate their pathogenesis. Quantitative shotgun proteomics can contribute to such objectives, allowing insights into the part of the genome translated into proteins and the connectivity of biochemical pathways and higher order assemblies of proteins at the subcellular level.Methodology/principal findingsWe examined protein profiles in cell lysate fractions of STEC strain 86-24 (serotype O157:H7), following growth in cell culture or bacterial isolation from intestines of infected piglets, in the context of functionally and structurally characterized biochemical pathways of E. coli. Protein solubilization in the presence of Triton X-100, EDTA and high salt was followed by size exclusion chromatography into the approximate M(r) ranges greater than 280 kDa, 280-80 kDa and 80-10 kDa. Peptide mixtures resulting from these and the insoluble fraction were analyzed by quantitative 2D-LC-nESI-MS/MS. Of the 2521 proteins identified at a 1% false discovery rate, representing 47% of all predicted E. coli O157:H7 gene products, the majority of integral membrane proteins were enriched in the high M(r) fraction. Hundreds of proteins were enriched in a M(r) range higher than that predicted for a monomer supporting their participation in protein complexes. The insoluble STEC fraction revealed enrichment of aggregation-prone proteins, including many that are part of large structure/function entities such as the ribosome, cytoskeleton and O-antigen biosynthesis cluster.SignificanceNearly all E. coli O157:H7 proteins encoded by prophage regions were expressed at low abundance levels or not detected. Comparative quantitative analyses of proteins from distinct cell lysate fractions allowed us to associate uncharacterized proteins with membrane attachment, potential participation in stable protein complexes, and susceptibility to aggregation as part of larger structural assemblies.

Project description:Cleavage and polyadenylation specificity factor 6 (CPSF6) is a human protein that binds HIV-1 capsid and mediates nuclear transport and integration targeting of HIV-1 preintegration complexes. Truncation of the protein at its C-terminal nuclear-targeting arginine/serine-rich (RS) domain produces a protein, CPSF6-358, that potently inhibits HIV-1 infection by targeting the capsid and inhibiting nuclear entry. To understand the molecular mechanism behind this restriction, the interaction between CPSF6-358 and HIV-1 capsid was characterized using in vitro and in vivo assays. Purified CPSF6-358 protein formed oligomers and bound in vitro-assembled wild-type (WT) capsid protein (CA) tubes, but not CA tubes containing a mutation in the putative binding site of CPSF6. Intriguingly, binding of CPSF6-358 oligomers to WT CA tubes physically disrupted the tubular assemblies into small fragments. Furthermore, fixed- and live-cell imaging showed that stably expressed CPSF6-358 forms cytoplasmic puncta upon WT HIV-1 infection and leads to capsid permeabilization. These events did not occur when the HIV-1 capsid contained a mutation known to prevent CPSF6 binding, nor did they occur in the presence of a small-molecule inhibitor of capsid binding to CPSF6-358. Together, our in vitro biochemical and transmission electron microscopy data and in vivo intracellular imaging results provide the first direct evidence for an oligomeric nature of CPSF6-358 and suggest a plausible mechanism for restriction of HIV-1 infection by CPSF6-358.IMPORTANCE After entry into cells, the HIV-1 capsid, which contains the viral genome, interacts with numerous host cell factors to facilitate crucial events required for replication, including uncoating. One such host cell factor, called CPSF6, is predominantly located in the cell nucleus and interacts with HIV-1 capsid. The interaction between CA and CPSF6 is critical during HIV-1 replication in vivo Truncation of CPSF6 leads to its localization to the cell cytoplasm and inhibition of HIV-1 infection. Here, we determined that truncated CPSF6 protein forms large higher-order complexes that bind directly to HIV-1 capsid, leading to its disruption. Truncated CPSF6 expression in cells leads to premature capsid uncoating that is detrimental to HIV-1 infection. Our study provides the first direct evidence for an oligomeric nature of truncated CPSF6 and insights into the highly regulated process of HIV-1 capsid uncoating.

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The FtsK-like motor TraB is a DNA-dependent ATPase that forms higher-order assemblies.

Publications

The FtsK-like motor TraB is a DNA-dependent ATPase that forms higher-order assemblies.

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