Figure 4

Mapping of UV-induced modifications detected by LC-MS/MS on to the dimeric crystal structures of the CRP-cAMP complex (PDB structure: 2wc2) and the CRP-cAMP-DNA complex (PDB structure: 1O3T). Panel A: Rendering of the sites and relative extents of modifications induced by UV-exposure in the presence of DNA. The two monomeric protein structures are indicated in grey and blue, with the DNA structure indicated in red. The extent of modification at individual amino acids is as indicated, with oxidation hotspots at Pro110/Met114, Pro160, Met163 and in the presence of DNA, Met189. Panels B and C: Rendering of the dimeric structures of the CRP-cAMP complex (panel B, PDB structure: 2wc2) and the CRP-cAMP-DNA complex (panel C, PDB structure: 1O3T). In panel C, the DNA strands are indicated in blue. The positions of the Trp13 (orange), Pro (yellow) and Met (red) residues in the two monomer structures is indicated. Comparison of panels B with C indicates the conformational changes in the CRP structure that occur on DNA binding. Panels D and E: Expansions of the dimer-interface region from panels B and C, indicating the Pro110 (yellow)-Met114 (red) pairs on the individual monomers and the increased proximity of these species at the dimer interface in the DNA-bound structure (panel E) compared to the non-bound (panel D). Modification at these residues, with conversion of Met114 to the sulfoxide and Pro110 to an alcohol are proposed to result in significant steric and electronic interactions that limit binding of UV-oxidised isolated cAMP-CRP to DNA (cf. Fig. 1A), and dissociation of cAMP-CRP from DNA when the bound complex is exposed to UV (cf. Fig. 1B). In panel E, the Trp13 residues (orange) are also indicated as these residues move closer to the Pro110-Met114 residues on DNA binding.