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

I. Tato, S. Zunzunegui, F. de la Cruz ^*, and E. Cabezon ^*

Departamento de Biología Molecular, Universidad de Cantabria, 39011 Santander, Spain

Communicated by Eugene W. Nester, University of Washington, Seattle, WA, April 25, 2005 (received for review January 12, 2005)

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. TrwBN70 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. TrwBN70 crystallographic structure revealed a hexamer with six equivalent subunits and a central channel. In this work, we characterize a DNA-dependent ATPase activity for TrwBN70. 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 TrwBN70–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.

molecular motor | DNA transfer

^* To whom correspondence may be addressed. E-mail: delacruz{at}unican.es or cabezone{at}unican.es.

© 2005 by The National Academy of Sciences of the USA

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