Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: alteration of DNA binding specificity through alteration of DNA kinking

Abstract

The catabolite activator protein (CAP) sharply bends DNA in the CAP-DNA complex, introducing a 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′-A 1A 2A 3T 4G 5T 6G 7A 8T 9C 10T 11-3′ (“primary kink”). CAP recognizes the base-pair immediately 5′ to the primary-kink site, T:A 6, through an “indirect-readout” mechanism involving sequence effects on the energetics of primary-kink formation. CAP recognizes the base-pair immediately 3′ to the primary-kink site, G:C 7, through a “direct-readout” mechanism involving formation of a hydrogen bond between Glu181 of CAP and G:C 7. Here, we report that substitution of the carboxylate side-chain of Glu181 of CAP by the one-methylene-group-shorter carboxylate side-chain of Asp changes DNA binding specificity at position 6 of the DNA half site, changing specificity for T:A 6 to specificity for C:G 6, and we report a crystallographic analysis defining the structural basis of the change in specificity. The Glu181→Asp substitution eliminates the primary kink and thus eliminates indirect-readout-based specificity for T:A 6. The Glu181→Asp substitution does not eliminate hydrogen-bond formation with G:C 7, and thus does not eliminate direct-readout-based specificity for G:C 7.