Structural basis of bifunctionality of Sinorhizobium meliloti Clr, a cAMP and cGMP receptor protein

In bacteria, Crp-Fnr superfamily transcription factors are the most ubiquitous receptor proteins of 3,5-cyclic adenosine monophosphate (cAMP) and 3,5-cyclic guanosine monophosphate (cGMP). The prototypic Escherichia coli CAP protein represents the main CRP subclass and is known to bind cAMP and cGMP, but to mediate transcription activation only in its cAMP-bound state. In contrast, both cyclic nucleotides mediate transcription activation by CRP subclass G protein Clr of Sinorhizobium meliloti. We present crystal structures of apo-Clr, and Clr*cAMP and Clr*cGMP bound to the core motif of the palindromic Clr DNA binding site (CBS). We show that both cyclic nucleotides shift ternary Clr*cNMP*CBS-DNA complexes to almost identical active conformations. Unlike the situation known for the E. coli CAP*cNMP complex, in the Clr*cNMP complex, the nucleobases of cGMP and cAMP are in the syn- and anti-conformation, respectively, allowing a shift to the active conformations in both cases. Isothermal titration calorimetry measured similar affinities of cAMP and cGMP binding to Clr in presence of CBS core motif DNA (KDcNMP 16 M). However, different affinities were determined in absence of this DNA (KDcGMP 24 M; KDcGMP 6 M). Sequencing of Clr co-immunoprecipitated DNA as well as Electrophoretic Mobility Shift and promoter-probe assays expanded the list of experimentally proven Clr-regulated promoters and CBS. This comprehensive set of CBS features conserved nucleobases, which are in agreement with the sequence readout through interactions of Clr amino acid residues with these nucleobases, as revealed by the Clr*cNMP*CBS-DNA crystal structures. IMPORTANCECyclic 3,5-adenosine monophosphate (cAMP) and cyclic 3,5-guanosine monophosphate (cGMP) are both long known as important nucleotide second messenger in eukaryotes. This is also the case for cAMP in prokaryotes, whereas a signaling role for cGMP in this domain of life has been recognized only recently. Catabolite repressor proteins (CRPs) are the most ubiquitous bacterial cAMP receptor proteins. Escherichia coli CAP, the prototypic transcription regulator of the main CRP subclass, binds both cyclic mononucleotides, but only the CAP*cAMP complex promotes transcription activation. In contrast, CRP subclass G proteins studied so far, are activated by cGMP, or both by cAMP and cGMP. Here, we report a structural analysis of the bifunctional cAMP- and cGMP-activatable Clr from Sinorhizobium meliloti, how binding of cAMP and cGMP shifts Clr to its active conformation, and the structural basis of its DNA binding site specificity.