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 molecular dynamics (MD) simulations of the CRP:(cAMP)(2) complex [García, A. E., and Harman, J. G. (1996) Protein Sci. 5, 62-71] showed repositioning of tyrosine 99 (Y99) to interact with the equatorial exocyclic oxygen atom of cAMP. To test the role of Y99 in cAMP-mediated CRP activation, Y99 was substituted with alanine (A) or phenylalanine (F). Cells that contained the WT or mutant forms of CRP induced beta-galactosidase in the presence of cAMP. Purified 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 CRP in cAMP binding cooperativity, in CRP:(cAMP)(1) complex binding to lacP DNA, in the formation of CRP:cAMP:RNAP complexes at lacP, or in CRP efficacy in mediating lacP activity in vitro. The apo-form of Y99A CRP was more sensitive to protease than the apo-form of either WT CRP or Y99F CRP. Whereas the WT or Y99F CRP:(cAMP)(1) complexes were cleaved by protease at hinge-region peptide bonds, the Y99A CRP:(cAMP)(1) complex was cleaved at peptide bonds 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 significantly greater than that measured for WT CRP. The results of this study show that tyrosine 99 contributes significant structural stability to the CRP dimer, specifically in stabilizing subunit association.
Baker
C H
CH
Theoretical Biology and Biophysics, Group T10, Mail Stop K710, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA.
Tomlinson
S R
SR
García
A E
AE
Harman
J G
JG
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites; D000242:Cyclic AMP / Q000737:chemistry / Q000378:metabolism; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits; D013816:Thermodynamics
D002373:Cyclic AMP Receptor Protein; D004269:DNA, Bacterial; D021122:Protein Subunits; D000242:Cyclic AMP