STRUCTURE-BASED REDESIGN OF COREPRESSOR SPECIFICITY OF THE ESCHERICHIA-COLI PURINE REPRESSOR BY SUBSTITUTION OF RESIDUE-190

Guanine or hypoxanthine, physiological corepressors of the Escherichia coli purine repressor (PurR), promote formation of the ternary PurR-c orepressor-operator DNA complex that functions to repress pur operon g ene expression. structure-based predictions on the importance of Arg19 0 in determining 6-oxopurine specificity and corepressor binding affin ity were tested by mutagenesis, analysis of in vivo function, and in v itro corepressor binding measurements. Replacements of Arg190 with Ala or Gin resulted in functional repressors in which binding of guanine and hypoxanthine was retained but specificity was relaxed to permit bi nding of adenine. X-ray structures were determined for ternary complex es of mutant repressors with purines (adenine, guanine, hypoxanthine, and 6-methylpurine) and operator DNA. These structures indicate that R 190A binds guanine, hypoxanthine, and adenine with nearly equal, albei t reduced, affinity in large part because of a newly made compensatory hydrogen bond between the rotated hydroxyl side chain of Ser124 and t he exocyclic 6 positions of the purines. Through direct and water-medi ated contacts, the R190Q protein binds adenine with a nearly 75-fold h igher affinity than the wild type repressor while maintaining wild typ e affinity for guanine and hypoxanthine. The results establish at the atomic level the basis for the critical role of Arg190 in the recognit ion of the exocyclic 6 position of its purine corepressors and the suc cessful redesign of corepressor specificity.