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 (A(2)) were 25.3(+/-0.5) A and - 0.39(+/-0.25) x 10(-4) ml mol g(-2), respectively. CO binding caused a slight increase in R-g (by 0.5 Angstrom) and a marked decrease in A(2) (by 5.09 x 10(-4) ml mol g(-2)). The observed decrease in A(2) points to higher attractive interactions between CO-bound CooA molecules in solution compared with CO-free CooA. Although the minor alternation of R-g 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 8degrees around the axis of 2-fold symmetry. (C) 2004 Elsevier Ltd. All rights reserved.