The Agrobacterium tumefaciens quorum-sensing transcriptional regulator TraR and its inducing ligand 3-oxo-octanoyl-l-homoserine lactone control conjugal transfer of the tumor-inducing plasmid, the primary virulence factor responsible for crown gall disease of plants. This regulatory system enables A. tumefaciens to express its conjugal transfer regulon preferentially at high population densities. TraR activity is antagonized by a second tumor-inducing plasmid-encoded protein designated TraM. TraM and TraR are thought to form an anti-activation complex that prevents TraR from recognizing its target DNA-binding sites. The formation and inhibitory function of the TraM-TraR anti-activation complex was analyzed using several different assays for protein-protein interaction, including surface plasmon resonance. The TraR-TraM complex forms readily in solution and is extremely stable (K(D) of 1-4 x 10(-9) m). Directed mutational analysis of TraM identified a number of amino acids that play important roles in the inhibition of TraR, clustering in two regions of the protein. Interestingly, several mutants were identified that proficiently bound TraR but were unable to inhibit its activity. This observation suggests a mechanistic separation between the initial assembly of the complex and conversion of TraR to an inactive form.