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Journal of Bacteriology, August 2003, p. 4371-4381, Vol. 185, No. 15
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.15.4371-4381.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

TraG-Like Proteins of Type IV Secretion Systems: Functional Dissection of the Multiple Activities of TraG (RP4) and TrwB (R388)

Max-Planck-Institut für Molekulare Genetik, Abteilung Lehrach, Dahlem, D-14195 Berlin, Germany

Received 4 April 2003/ Accepted 30 April 2003

TraG-like proteins are essential components of type IV secretion systems. During secretion, TraG is thought to translocate defined substrates through the inner cell membrane. The energy for this transport is presumably delivered by its potential nucleotide hydrolase (NTPase) activity. TraG of conjugative plasmid RP4 is a membrane-anchored oligomer that binds RP4 relaxase and DNA. TrwB (R388) is a hexameric TraG-like protein that binds ATP. Both proteins, however, lack NTPase activity under in vitro conditions. We characterized derivatives of TraG and TrwB truncated by the N-terminal membrane anchor (TraG2 and TrwB1) and/or containing a point mutation at the putative nucleotide-binding site (TraG2K187T and TraGK187T). Unlike TraG and TrwB, truncated derivatives behaved as monomers without the tendency to form oligomers or aggregates. Surface plasmon resonance analysis with immobilized relaxase showed that mutant TraGK187T was as good a binding partner as the wild-type protein, whereas truncated TraG monomers were unable to bind relaxase. TraG2 and TrwB1 bound ATP and, with similar affinity, ADP. Binding of ATP and ADP was strongly inhibited by the presence of Mg²⁺ or single-stranded DNA and was competed for by other nucleotides. Compared to the activity of TraG2, the ATP- and ADP-binding activity of the point mutation derivative TraG2K187T was significantly reduced. Each TraG derivative bound DNA with an affinity similar to that of the native protein. DNA binding was inhibited or competed for by ATP, ADP, and, most prominently, Mg²⁺. Thus, both nucleotide binding and DNA binding were sensitive to Mg²⁺ and were competitive with respect to each other.

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