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

The catabolite activator protein (CAP) sharply bends DNA in the CAP-DNA complex, introducing a DNA kink, with a roll angle of similar to 40 degrees and a twist angle of similar to 20 degrees, between positions 6 and 7 of the DNA half-site, 5'-A(1)A(2)A(3)T(4)G(5)T(6)G(7)A(8)T(9)C(10)T(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 sidechain 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. (C) 2001 Academic Press.