cAMP-mediated effects on CRP subunit interactions

Cyclic 3':5'-adenosine monophosphate (cAMP) receptor protein (CRP) of Escherichia coli is a dimeric 47 kDa protein composed of identical subunits. Each subunit can bind cyclic nucleotide. CRP exhibits negative cooperativity in binding cAMP and the current model supports three different CRP conformers: apo-CRP the CRP:(cAMP)^1 complex, and the CRP: cAMP)^2 complex. In the presence of cAMP. CRP binds to specific DNA sequences located upstream of a number of E. coli promoters. The CRP:(c.AMP)^1 complex regulates transcription from these promoters. The conformational changes that activate CRP are not completely understood. Weber and Steitz (J. Mol.Biol. 1987) predicted that upon binding cAMP. CRP subunits are likeh to reorient. To test this hypothesis amino acids along the CRP C helix were substituted with cysteine to allow intermolecular disulfide bond formation. Disulfide crosslinked CRP's were analyzed to confirm the presence of intermolecular positional pair disulfide bonds. The distribution of these disulfide bonds was analyzed over a wide range of cAMP concentrations. These experiments show that while there were only modest cAMPdependent changes in disulfide bond distribution, the C-proximal end of the C helix exhibited a surprising degree of flexibility.

During the course of this study, leucine (L) at position 124, located in the cyclic nucleotide binding pocket of CRP, was shown to be important in CRP activation. The substitution of L124 with alanine (A) decreased the affinity of CRP for the first molecule of cAMP by about one order of magnitude and decreased negative cooperativity in cAMP binding by 55-fold. In the presence of cAMP cells containing L124A CRP did not induce B-galactosidase in vivo. In vitro studies showed cAMP binding to L124A failed to produce conformational changes in the CRP. The hydrophobic side chain bulk of L124, which packs against cAMP, appears important in cAMP binding and in CRP activation.

Finally, an assay for monitoring CRP subunit exchange was developed to assess the affect of amino acid substitutions at CRP position 99. The results of this study provide conclusive evidence that tyrosine 99 plays an important role in stabilizing the association of CRP subunits.