CyclicAMP receptor protein (CRP) regulates transcription
of numerous genes in Escherichia coli. Both cAMP
and cGMP bind CRP, but only cAMP induces conformational
changes that dramatically increase the specific DNA binding
activity of the protein. We have shown previously that
our protein footprinting 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 mapped and quantitatively analyzed
by protein footprinting using iron complexed to diethylenetriaminepentaacetic
acid ([Fe-DTPA]2−), iron complexed
to ethylenediaminediacetic acid ([Fe-EDDA]),
iron complexed to desferrioxamine mesylate ([Fe-HDFO]+),
and copper complexed to o-phenanthroline ([(OP)2Cu]+)
as proteases. These chemical proteases differ in size,
charge, and hydrophobicity. 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 α-helix, the interdomain hinge, and the ends of the
C α-helix, which is involved in intersubunit interactions.
[Fe-EDDA] and [Fe-DTPA]2−
also detected reductions in cleavage in the D and E α-helices,
which are involved in DNA recognition. Cleavage by [Fe-HDFO]+
of CRP-cAMP detected hypersensitivities in β-strand
8, the B α-helix, as well as in parts of the F and
C α-helices. [Fe-HDFO]+ also detected
protections from cleavage in β-strands 4 to 5 and their
intervening loop, β-strand 7, which is part of the
nucleotide binding pocket, as well as in the D and E α-helices.
Cleavage by [(OP)2Cu]+
of CRP-cAMP detected hypersensitivities in β-strands
9 and 11 as well as in the D and E α-helices. [(OP)2Cu]+
also detected protections in the C α-helix , the interdomain
hinge, and β-strands 2–7. Binding of cGMP to
CRP resulted in changes in susceptibility to cleavage only
by [(OP)2Cu]+, which detected
minor protections in β-strands 3–7, the interdomain
hinge, and the C α-helix. These results show that binding
of cAMP causes structural changes in CRP in the nucleotide
binding domain, the interdomain hinge, the DNA binding
domain, and regions involved in intersubunit interaction.
Structural changes induced by binding of cGMP appear to
be very minor and confined to the nucleotide binding domain,
the interdomain hinge, and regions involved in intersubunit
interaction. Use of different cleaving agents in protein
footprinting seems to give a more detailed picture of structural
changes than the use of a single protease alone.