Structure-Based Redesign of Corepressor Specificity of the Escherichia coli Purine Repressor by Substitution of Residue 190

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• 作者：Fu Lu ; Maria A. Schumacher ; Dennis N. Arvidson ; Andreas Haldimann ; Barry L. Wanner ; Howard Zalkin ; and Richard G. Brennan
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：January 27, 1998
• 年：1998
• 卷：37
• 期：4
• 页码：971 - 982
• 全文大小：202K
• 年卷期：v.37,no.4(January 27, 1998)
• ISSN：1520-4995

文摘

Guanine or hypoxanthine, physiological corepressorsof the Escherichia coli purine repressor(PurR), promote formation of the ternary PurR-corepressor-operatorDNA complex that functions torepress pur operon gene expression. Structure-basedpredictions on the importance of Arg190 indetermining 6-oxopurine specificity and corepressor binding affinitywere tested by mutagenesis, analysisof in vivo function, and in vitro corepressor binding measurements.Replacements of Arg190 with Alaor Gln resulted in functional repressors in which binding of guanineand hypoxanthine was retained butspecificity was relaxed to permit binding of adenine. X-raystructures were determined for ternarycomplexes of mutant repressors with purines (adenine, guanine,hypoxanthine, and 6-methylpurine) andoperator DNA. These structures indicate that R190A binds guanine,hypoxanthine, and adenine withnearly equal, albeit reduced, affinity in large part because of a newlymade compensatory hydrogen bondbetween the rotated hydroxyl side chain of Ser124 and the exocyclic 6positions of the purines. Throughdirect and water-mediated contacts, the R190Q protein binds adeninewith a nearly 75-fold higher affinitythan the wild type repressor while maintaining wild type affinity forguanine and hypoxanthine. Theresults establish at the atomic level the basis for the critical roleof Arg190 in the recognition of theexocyclic 6 position of its purine corepressors and the successfulredesign of corepressor specificity.