Escherichia coli purine repressor: Redesign corepressor specificity and identify key residues involved in allosteric transition

Fu Lu, Purdue University

Abstract

The purpose of the present study was to investigate the corepressor binding specificity of Escherichia coli PurR and identify key amino acid residues and interactions required for the allosteric transition induced by corepressor binding. Structure-based predictions on the importance of Arg190 and Glu222 in determining corepressor binding specificity at exocyclic 6- and 2-positions, respectively, were tested by mutagenesis, analysis of in vivo function, and in vitro 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 binding of adenine. The Glu222 to Ala or Gin replacements reduced the relative specificity of repressors for Gua over hypoxanthine from 7 to 2 fold. The E222K mutant bound guanine, hypoxanthine and xanthine with less than 2 fold difference in affinity. Thus, corepressor specificity at exocyclic 6- and 2-positions was successful redesigned. Key residues and interactions involved in the corepressor modulated allosteric transition were investigated by site-directed mutagenesis. The wild type PurR is in the open inactive conformation in the absence of corepressor. Results from a series of biochemical and biophysical analyses support the conclusion that the apo mutant repressors, W147A and E70A, were in the closed active conformational state in the absence of corepressor. This finding is consistent with the proposal that Trp147 plays a key role in stabilizing the open conformation as a structural surrogate of corepressor and indicates that Glu70 plays an important role in the allosteric transition process in the corepressor binding domain. Mutational analysis was used to identify an Arg 115-Ser46’ interdomain intersubunit hydrogen bond that is necessary for transmitting the allosteric transition in the corepressor binding domain to the DNA binding domain. R115A and S46G PurR mutants were defective in DNA binding in vitro and repressor function in vivo although corepressor binding was identical to the wild type. These results establish that the hydrogen bond between the NH2 of Arg 115 and the main chain CO of Ser 46’ plays a critical role in interdomain signalling.

Degree

Ph.D.

Advisors

Zalkin, Purdue University.

Subject Area

Molecular biology|Microbiology|Biochemistry

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