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.

Introduction

Sequence-specific DNA-binding proteins primarily recognize DNA sites through “direct readout,” involving hydrogen bonds and van der Waals interactions with DNA base-pairs.1, 2, 3 However, sequence-specific DNA-binding proteins also can recognize DNA sites through “indirect readout,” without direct contact with DNA base-pairs.2, 3, 4, 5, 6, 7, 8, 9 Indirect readout is less well characterized than direct readout. Mechanisms for indirect readout include, in principle, water-mediated hydrogen bonds, sequence effects on DNA conformation, and sequence effects on susceptibility to DNA deformation.

One clear example of indirect readout involves the Escherichia coli catabolite activator protein (CAP; also known as the cAMP receptor protein, CRP), which recognizes the consensus DNA half-site 5′-A₁A₂A₃T₄G₅T₆G₇A₈T₉C₁₀T₁₁-3′.10, 11, 12 CAP makes no direct contacts to the T:A base-pair at position 6 of the consensus DNA half-site;13, 14, 15 nevertheless, CAP displays strong specificity for T:A at position 6, preferring T:A over A:T, C:G, and G:C by 1.5, 1.4, and 1.4 kcal/mol per half-site, respectively.¹² In the CAP–DNA complex, CAP sharply bends DNA, typically introducing a DNA kink, with a roll angle of ∼40° and a twist angle of ∼20°, between positions 6 and 7 (“primary kink”).13, 14, 15, 16, 17, 18 In the consensus half-site, positions 6 and 7 are part of a T:A/G:C base-pair step, a base-pair step that is associated with high roll angles19, 20, 21, 22, 23 and that has high susceptibility to roll deformation.24, 25, 26, 27 It has been proposed that the specificity for T:A at position 6 is a consequence of the primary kink between positions 6 and 7, and of the effects of the T:A/G:C step on DNA deformation and/or DNA deformability.¹⁴ Two specific mechanisms for discrimination against non-consensus base-pairs at position 6 have been considered: (i) altered DNA deformation, wherein non-consensus base-pairs at position 6 partly or fully preclude formation of the primary kink and thus partly or fully preclude formation of CAP–DNA interactions distal to the primary kink (e.g. interactions with DNA phosphates at positions −3 through 5); and (ii) altered energetic susceptibility to DNA deformation, wherein both consensus and non-consensus base-pairs at position 6 permit formation of the primary kink, but non-consensus base-pairs impose a higher energetic cost for formation of the primary kink.

In previous work, in a first effort to understand this example of indirect readout, we determined crystal structures of complexes of CAP with a DNA site containing the non-consensus base-pair C:G at position 6.²⁸ We observed, in each of two crystal forms, that complexes containing the consensus base-pair T:A or the non-consensus base-pair C:G exhibited essentially identical overall DNA bend angles and local geometries of DNA kinking. We inferred that discrimination between the consensus base-pair T:A or the non-consensus base-pair C:G does not involve differences in the deformation of DNA, but, rather, solely differences in the energetics of DNA kinking.

Here, to understand further this example of indirect readout, we have extended the analysis to complexes of CAP with DNA sites containing all possible base-pairs at position 6. The results show that DNA kinking can occur with any base at position 6, but reveal qualitative differences in DNA kinking with pyrimidine-purine steps at positions 6-7 versus with purine-purine steps at positions 6-7 and thus suggest differences in mechanisms of discrimination against non-consensus pyrimidine-purine steps versus discrimination against non-consensus purine-purine steps.