The role of the interdomain B linker in the activation of the XylR proteinof Pseudomonas putida

In the presence of toluene and other structural analogues, the enhancer bin ding protein XylR activates the sigma (54) promoter Pu of the TOL (toluene degradation) plasmid pWW0 of Pseudomonas putida. Introduction of amino acid changes Val-219Asp and Ala-220Pro, which enter a proline kink at the inter domain region (B linker) between the A (signal reception) module and the ce ntral portion of XylR, originated a protein with unforeseen properties. The se included a minor ability to activate Pu in the absence of aromatic effec tors, a much higher responsiveness to m-xylene and a significant response t o a large collection of aromatic inducers. Such changes could not be attrib uted to variations in XylR expression levels or to the fortuitous creation of a novel promoter, but to a genuine change in the properties of the activ ator. Structural predictions suggested that the mutation entirely disrupted an otherwise probable coiled-coil structure. A second directed mutant with in the same region consisting of a major replacement of amino acids A220-N2 21 by the peptide HHHR produced an even more exacerbated phenotype. These d ata support a model in which the linker B region influences the effector pr ofile by modifying at a distance the operative shape of the effector pocket and fixing the protein in an intermediate step of the activation process.