Biochemical characterization of CgrA, a novel cGMP-binding CRP homolog from Rhodospirillum centenum

Bacterial physiological status is controlled in part by a signal transduction network of small molecules that integrate information from both their extracellular and intracellular environment of the cell. Several intracellular signaling pathways are controlled by cyclic nucleotides that integrate information from outside to and inside of the cell. Under proper circumstances, these small molecules elicit appropriate responses by acting as a signal to a receptor molecule that contains an output domain. The well-characterized family of proteins that sense and bind small nucleotide molecules is the E. coli CRP family of transcriptional regulators. In my doctoral thesis, I have characterized a novel CRP homolog, called CgrA that binds cyclic guanosine monophosphate (cGMP) as a signal to trigger Rhodospirillum centenum to induce metabolically dormant cysts that survive harsh environmental conditions. In this thesis, I have utilized a combination of biochemical and biophysical studies to show that CgrA binds cGMP in a manner similar to how E. coli CRP binds cAMP. I also used fluorescence anisotropy to show that cGMP nucleotide binding by CgrA stimulates recognition of specific DNA sequences in a manner that is similar to that of CRP binding to DNA in the presence of cAMP. This work thus highlights and contrasts the mechanism of gene expression by R. centenum CgrA with that of the well characterized E. coli CRP. To obtain an understanding of how CgrA might perceive cGMP and differentiate it from other cyclic nucleotides, I also used a combination of homology modeling and site-directed mutagenesis using E. coli CRP-cAMP crystal structure as a reference molecule. Mutagenesis studies have identified Arg100 as an important residue in establishing hydrogen bond formation with the negative phosphate backbone of the ribose moiety of the cGMP and thus an Arg100Leu mutation completely abolishes binding to any cyclic nucleotide. Additional mutations however, failed to identify any amino acid residues that confer cGMP specificity over that of cAMP. Thus, although both proteins are homologues, they share remote similarity in their core ligand binding pocket suggesting that CgrA binds cGMP in a manner that is distinct from cAMP binding by CRP. I also undertook crystallization trials with full-length native CgrA that has yielded two promising crystal growth conditions one of which closely resembles the crystallization conditions of Clp, a CRP homolog from Xanthomonas campestris.