Indirect Readout of DNA Sequence at the Primary-kink Site in the CAP–DNA Complex: Recognition of Pyrimidine-Purine and Purine-Purine Steps

The catabolite activator protein (CAP) bends DNA in the CAP–DNA complex, typically introducing a sharp DNA kink, with a roll angle of ∼40° and a twist angle of ∼20°, between positions 6 and 7 of the DNA half-site, 5′-A1A2A3T4G5T6G7A8T9C10T11-3′ (“primary kink”). In previous work, we showed that CAP recognizes the nucleotide immediately 5′ to the primary-kink site, T6, through an “indirect-readout” mechanism involving sequence effects on energetics of primary-kink formation. Here, to understand further this example of indirect readout, we have determined crystal structures of CAP–DNA complexes containing each possible nucleotide at position 6. The structures show that CAP can introduce a DNA kink at the primary-kink site with any nucleotide at position 6. The DNA kink is sharp with the consensus pyrimidine-purine step T6G7 and the non-consensus pyrimidine-purine step C6G7 (roll angles of ∼42°, twist angles of ∼16°), but is much less sharp with the non-consensus purine-purine steps A6G7 and G6G7 (roll angles of ∼20°, twist angles of ∼17°). We infer that CAP discriminates between consensus and non-consensus pyrimidine-purine steps at positions 6-7 solely based on differences in the energetics of DNA deformation, but that CAP discriminates between the consensus pyrimidine-purine step and non-consensus purine-purine steps at positions 6-7 both based on differences in the energetics of DNA deformation and based on qualitative differences in DNA deformation. The structures further show that CAP can achieve a similar, ∼46° per DNA half-site, overall DNA bend through a sharp DNA kink, a less sharp DNA kink, or a smooth DNA bend. Analysis of these and other crystal structures of CAP–DNA complexes indicates that there is a large, ∼28° per DNA half-site, out-of-plane component of CAP-induced DNA bending in structures not constrained by end-to-end DNA lattice interactions and that lattice contacts involving CAP tend to involve residues in or near biologically functional surfaces.