The induction mechanism of the flavonoid‐responsive regulator FrrA

Abstract

Bradyrhizobium diazoefficiens, a bacterial symbiont of soybean and other leguminous plants, enters a nodulation-promoting genetic programme in the presence of host-produced flavonoids and related signalling compounds. Here, we describe the crystal structure of an isoflavonoidresponsive regulator (FrrA) from Bradyrhizobium, as well as cocrystal structures with inducing and noninducing ligands (genistein and naringenin, respectively). The structures reveal a TetR-like fold whose DNAbinding domain is capable of adopting a range of orientations. A single molecule of either genistein or naringenin is asymmetrically bound in a central cavity of the FrrA homodimer, mainly via C-H contacts to the psystem of the ligands. Strikingly, however, the interaction does not provoke any conformational changes in the repressor. Both the flexible positioning of the DNA-binding domain and the absence of structural change upon ligand binding are corroborated by small-angle X-ray scattering (SAXS) experiments in solution. Together with a model of the promoter-bound state of FrrA our results suggest that inducers act as a wedge, preventing the DNA-binding domains from moving close enough together to interact with successive positions of the major groove of the palindromic operator.

Crystal structures of free and ligand‐bound forms of the TetR/AcrR‐like regulator SCO3201 from Streptomyces coelicolor suggest a novel allosteric mechanism

“…The conformational changes that we observe in SCO3201 are ultimately caused by a slight expansion of the binding pocket, a direct consequence of the bulkiness of the inducer. This effect should be virtually independent of the precise binding specificity of the cavity, a phenomenon that we have also observed for the induction mechanisms of TetR(D) [32] and FrrA [25]. As noted before, modularity of ligand recognition and allostery is expected to result in a considerable degree of evolutionary flexibility, allowing existing transcriptional regulators to mutate and adapt to new inducers and regulatory circuits while their induction mechanism remains intact [32].…”

“…Intriguingly, the compact arrangement of the two HTH-motifs, with their recognition helices at less than 25 A from each other, would preclude binding to consecutive instances of the major groove on one face of the promoter. This DNA-binding mode, which is a hallmark of the TetR/AcrR family [10], requires an a3-a3 distance very close to 35…”

“…Although DNA binding by members of the TetR/AcrR family is typically accompanied by appreciable structural changes in the regulatory domain [ 10 ], all currently known cases involve DNA‐binding domains that are far apart in the free repressor and need to be brought into closer proximity to enable promoter binding. Inducers can then act simply by preventing such rearrangements [ 25 ]. Strikingly, this is the exact opposite of what we seem to observe for SCO3201.…”

“…It is currently unclear whether or not the SCO3201 dimer is able to interact with a second inducer molecule, which might further enlarge the physical distance between the HTH motifs and lead to a complex transcriptional response to increasing ligand concentrations. It should be noted, however, that many TetR/AcrR‐like repressors only bind a single ligand molecule per homodimer [ 10 ], either in a central cavity around the symmetry axis of the dimer, as in FrrA [ 25 ], or in one of several available pockets within the individual subunits, for example in QacR [ 30 ] and TtgR [ 31 ].…”