TrwB, the coupling protein involved in DNA transport during bacterial conjugation, is a DNA-dependent ATPase.
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. TrwBDeltaN70 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. TrwBDeltaN70 crystallographic structure revealed a hexamer with six equivalent subunits and a central channel. In this work, we characterize a DNA-dependent ATPase activity for TrwBDeltaN70. 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 TrwBDeltaN70-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 F1-ATPase, lead us to propose a mechanism for TrwB as a DNA-translocating motor.
Tato
I
I
Departamento de Biología Molecular, Universidad de Cantabria, 39011 Santander, Spain.
Zunzunegui
S
S
de la Cruz
F
F
Cabezon
E
E
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001692:Biological Transport; D002850:Chromatography, Gel; D003227:Conjugation, Genetic / Q000502:physiology*; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005799:Genes, Dominant / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008958:Models, Molecular; D020836:Protein Structure, Quaternary; D017384:Sequence Deletion / Q000235:genetics
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C403125:trwB protein, E coli; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; D004265:DNA Helicases