Protein-ligand interaction: Grafting of the uridine-specific determinants from the CytR regulator of Salmonella typhimurium to Escherichia coli CytR

Members of the LacI family of transcriptional repressors respond to the pre sence of small effector molecules. The binding of the ligands affect the pr oteins ability to repress transcription by stabilizing a conformation that, in most cases, is unfavorable for high-affinity DNA binding. The CytR anti -activator diverges from the other family members by relying on the coopera tive DNA binding with the global regulator CRP. The inducers of CytR do not affect CytR-DNA binding per se, but alleviate repression by interrupting p rotein-protein interactions between the two regulators. Here, we have studi ed of the CytR-inducer interaction by exploring a discrepancy in the induce r response observed for the homologous CytR regulators of Escherichia coli and Salmonella typhimurium. CytR of S. typhimurium (CytR(St)) appears to re spond to the presence of both uridine and cytidine nucleosides, whereas E. coli CytR (CytR(Ec)) responds to cytidine only. We have used a combination of genetic and structural modeling studies to provide detailed information regarding the nature of this discrepancy. By analysis of hybrid CytR protei ns followed by site-directed mutagenesis, we have successfully transferred the specificity determinants for uridine from CytR(St) to CytR(Ec), reveali ng that serine substitutions of only two residues (G131 and A152) in CytR(E c) is required to make CytR(Ec) sensitive to uridine. In addition, by emplo ying a genetic screen for induction of defective mutants, we have identifie d four amino acid residues in CytR(St) that appear to be important for the response to uridine. The implications of these findings for the understandi ng of the ligand binding and induction of CytR are discussed in the context of the structural knowledge of CytR and homologous protein-ligand complexe s. (C) 1999 Academic Press.