The role of positions 99 and 127 in cAMP-mediated activation of CRP
Baker, Cheryl H.
MetadataShow full item record
The cycHc 3':5'-adenosine monophosphate (cAMP) receptor protein (CRP) of Escherichia coli hinds cycHc nucleotides. When complexed with cAMP, CRP binds to specific DNA sequences located upstream of a number of promoters leading to the formation of active transcription complexes composed of CRP, cAMP, DNA, and RNA polymerase (RNAP). Molecular dynamics (MD) simulations were performed on CRP:(cAMP)2 structure. Analysis of amino acid residue proximities to cAMP in the MD-CRP:(cAMP)2 complex confirmed that known protein/Ugand contacts were maintained during the simidation and revealed the repositioning of tyrosine at position 99 (Y99) to interact with cAMP. To determine the vahdity of the MDpredicted Y99:cAMP interaction the crp gene was mutated to replace the Y99 codon with either an alanine (A) or a phenylalanine (F) codon. The mutant genes were expressed and characterized in vivo. Cells that contained wildtype (WT), alanine (Y99A), or phenylalanine (Y99F) CRP expressed pgalactosidase only when cultured in the presence of cAMP. Those cultures that contained Y99A or Y99F CRP showed cAMP-dependent inhibition of cell growth. In vitro studies showed that purified WT, Y99A and Y99F CRP found sequentially two molecules of cAMP, exhibited negative cooperativity in cAMP binding and activated the lactose promoter (lacP)in the presence of cAMP. These data, when coupled with the characteristics of cAMPS(Rp)binding to and effect on WT CRP, are consistent with a Y99:R82:cAMP interaction observed for the CRP:(cAMP)2 complex. In addition to the Y99 studies, the DNA binding and lacP activation characteristics of mutant forms of CRP (cysteine [C], glycine [G], isoleucine [I] or serine [S] for threonine [T]) at position 127 were investigated. The results demonstrated that allosteric changes important for cAMP-mediated CRP DNA binding is differentially affected by amino acid substitution at position 127. However, cAMP-dependent interaction of CRP with RNAP observed regardless of the amino acid at position 127. This study further investigates the characteristics of these mutants with respect to lacP DNAand cAMP-binding. The results lead to the conclusion that threonine 127 plays an important role in transduction of the signal from the CRP cyclic nucleotide binding pocket that promotes proper orientation of the DNAbinding helices and only a minor, if any, role in the functional exposure of the CRP RNAP interaction domain.