Activation Mechanisms of Transcriptional Regulator CooA Revealed by Small-angle X-ray Scattering

CooA, a heme-containing transcriptional activator, binds CO to the heme moiety and then undergoes a structural change that promotes the specific binding to the target DNA. To elucidate the activation mechanism coupled to CO binding, we investigated the CO-dependent structural transition of CooA with small-angle X-ray scattering (SAXS). In the absence of CO, the radius of gyration (Rg) and the second virial coefficient (A2) were 25.3(±0.5) Å and −0.39(±0.25)×10−4 ml mol g−2, respectively. CO binding caused a slight increase in Rg (by 0.5 Å) and a marked decrease in A2 (by 5.09×10−4 ml mol g−2). The observed decrease in A2 points to higher attractive interactions between CO-bound CooA molecules in solution compared with CO-free CooA. Although the minor alternation of Rg rules out changes in the overall structure, the marked change in the surface properties points to a CO-induced conformational transition. The experimental Rg and SAXS curves of the two states did not agree with the crystal structure of CO-free CooA. We thus simulated the solution structures of CooA based on the experimental data using rigid-body refinements as well as low-resolution model reconstructions. Both results demonstrate that the hinge region connecting the N-terminal heme domain and C-terminal DNA-binding domain is kinked in CO-free CooA, so that the two domains are positioned close to each other. The CO-dependent structural change observed by SAXS corresponds to a slight swing of the DNA-binding domains away from the heme domains coupled with their rotation by about 8° around the axis of 2-fold symmetry.