Analysis of interaction between GbdR, a choline metabolism regulator, with DNA and RNA polymerase

BEASSONI, PR; PRIMO, E; LISA, AT

Torremolinos

Conferencia; IV International Conference on Environmental, Industrial and Applied Microbiology - BioMicroWorld 2011; 2011

Formatex Research Center

Many genes encoding proteins involved in the pathogenesis of Pseudomonas aeruginosa such as phospholipase C and phosphorylcholine phosphatase are regulated by GbdR, a protein encoded by the gene PA5380. This regulator also activates the genes of choline catabolism. In this work we studied the relationship between structure and function of P. aeruginosa GbdR. For this, gbdR was cloned, the recombinant protein was obtained and the interaction with DNA was demonstrated by gel shift assays. GbdR is homologue with MarA, a transcriptional activator of the AraC family that activates at least 24 promoters of E. coli. The structure of MarA was determined by NMR in complex with a synthetic DNA and the C-terminal domain of the a subunit of RNA polymerase (a-CTD-RNAP) (PDB: 1XS9). Using threading techniques, we have modeled P. aeruginosa GbdR using as template the atomic coordinates of MarA of E. coli (PDB:1XS9) for the C-terminal domain and, the protein Atu0886 from A. tumefasciens for N-terminal domain. The obtained model, evaluated using the assessment tools available in Swiss-Model workspace, indicated the presence of two DNA binding motifs helix-turn-helix (HTH), suggesting that these are the critical regions of GbdR that interact with DNA. To understand the function of the DNA-regulator-polymerase complex, the a-CTD domain of P. aeruginosa RpoA was also modeled by comparative modeling in Swiss-Model workspace using atomic coordinated of the same protein of E. coli (PDB:1XS9). The complex a-CTD-RNAP-GbdR-DNA was obtained and we suggest that the two a-helix of GbdR that interact with DNA are: 254RRQLERLFQKYL265 and 304TPHFSKCYREYF315. In addition, we also propose that the regions interacting in the protein-protein interface are: 19V, 20R, 23N, 24C, 49N, and 84D for RNAP; 219L, 228E, 231A, 232L, and 235A for GbdR. In conclusion, we were able to identify the residues of GbdR that might be involved in the interaction with DNA and with RNAP. So, with all these bioinformatic results we have opened a way to discover the relevance of these regions which will be tested experimentally by site-directed mutations.