Mapping cyclic nucleotide-induced conformational changes in cyclicAMP receptor protein by a protein footprinting technique using different chemical proteases

CyclicAMP receptor protein (CRP) regulates transcription of numerous genes in Escherichia coli. Both cAMP and cGMP bind CRP, but only cAMP induces con formational changes that dramatically increase the specific DNA binding act ivity of the protein. We have shown previously that our protein footprintin g technique is sensitive enough to detect conformational changes in CRP by cAMP [Baichoo N, Heyduk T. 1997. Biochemistry 36:10830-10836]. In this work , conformational changes in CRP induced by cAMP and cGMP binding were mappe d and quantitatively analyzed by protein footprinting using iron complexed to diethylenetriaminepentaacetic acid ([Fe-DTPA](2-)), iron complexed to et hylenediaminediacetic acid ([Fe-EDDA]), iron complexed to desferrioxamine m esylate ([Fe-HDFO](+)), and copper complexed to o-phenanthroline ([(OP)(2)C u](+)) as proteases. These chemical proteases differ in size, charge, and h ydrophobicity. Binding of cAMP to CRP resulted in changes in susceptibility to cleavage by all four proteases. Cleavage by [Fe-EDDA]and [Fe-DTPA](2-) of CRP-cAMP detected hypersensitivities in the DNA-binding F alpha-helix, t he interdomain hinge, and the ends of the C alpha-helix, which is involved in intersubunit interactions. [Fe-EDDA] and [Fe-DTPA](2-) also detected red uctions in cleavage in the D and E alpha-helices, which are involved in DNA recognition Cleavage by [Fe-HDFO](+) of CRP-cAMP detected hypersensitiviti es in beta-strand 8, the B alpha-helix, as well as in parts of the F and C alpha-helices. [Fe-HDFO](+) also detected protections from cleavage in beta -strands 4 to 5 and their intervening loop, beta-strand 7, which is part of the nucleotide binding pocket, as well as in the D and E alpha-helices. Cl eavage by [(OP)(2)Cu](+) of CRP-cAMP detected hypersensitivities in beta-st rands 9 and 11 as well as in the D and E alpha-helices. [(OP)(2)Cu](+) also detected protections in the C alpha-helix, the interdomain hinge, and beta -strands 2-7. Binding of cGMP to CRP resulted in changes in susceptibility to cleavage only by [(OP)(2)Cu](+), which detected minor protections in bet a-strands 3-7, the interdomain hinge, and the C alpha-helix. These results show that binding of cAMP causes structural changes in CRP in the nucleotid e binding domain, the interdomain hinge, the DNA binding domain, and region s involved in intersubunit interaction. Structural changes induced by bindi ng of cGMP appear to be very minor and confined to the nucleotide binding d omain, the interdomain hinge, and regions involved in intersubunit interact ion. Use of different cleaving agents in protein footprinting seems to give a more detailed picture of structural changes than the use of a single pro tease done.