Amino acid substitution at position 99 affects the rate of CRP subunit exchange

We investigated the characteristics of CRP having amino acid substitutions at position 99. Analysis of amino acid residue proximity to cAMP in molecul ar dynamics (MD) simulations of the CRP: (cAMP)(2) complex [Garcia, A. E., and Harman, J. G. (1996) Protein Sci. 5, 62-71] showed repositioning of tyr osine 99 (Y99) to interact with the equatorial exocyclic oxygen atom of cAM P. To test the role of Y99 in cAMP-mediated CRP activation, Y99 was substit uted with alanine (A) or phenylalanine (F). Cells that contained the WT or mutant forms of CRP induced beta -galactosidase in the presence of cAMP. Pu rified WT, Y99A, and Y99F CRP showed only a 3- to 4-fold difference in cAMP affinity. There were no apparent differences between the three forms of CR P in cAMP binding cooperativity, in CRP:(cAMP), complex binding to lacP DNA , in the formation of CRP:cAMP:RNAP complexes at lacP, or in CRP efficacy i n mediating lacP activity in vitro. The apo-form. of Y99A CRP was more sens itive to protease than the apo-form of either WT CRP or Y99F CRP. Whereas t he WT or Y99F CRP:(cAMP), complexes were cleaved by protease at hinge-regio n peptide bonds, the Y99A CRP:(cAMP)(1) complex was cleaved at peptide bond s located at the subunit interface. The rates of subunit exchange for Y99A CRP, both in the apo-form and in a 1:1 complex with cAMP, were significantl y greater than that measured for WT CRP. The results of this study show tha t tyrosine 99 contributes significant structural stability to the CRP dimer , specifically in stabilizing subunit association.