Elucidation of the regulatory mechanisms of the diguanylate cyclases PleD, DgcA and DgcB by structural and biophysical analysis

The ubiquitous bacterial second messenger bis-(3’-5’)-cycic di-guanosine monophosphate (c-di-GMP) turns out to be the key regulator of the antagonistic processes: motility of individual cells on the one hand and persistence of a bacterial population in biofilms on the other. The biosynthesis of c-di-GMP by consumption of two molecules GTP is performed by diguanylate cyclases (DGCs). DGCs consist of catalytic GGDEF domains in combination with a plethora of N-terminal, environment sensing regulatory domains. The previously elucidated crystal structure of the DGC PleD from C. crescentus has shown that the catalytic GGDEF domain shares its fold with the well studied adenylate cyclases and DNA polymerases. The ability of the GGDEF domain to bind only one GTP molecule requires formation of a complete joint-active site formed by two GGDEF domains. Therefore, DGCs have to form dimers to be enzymatically active. DGCs widely exploit environment-sensing domains for the regulation of their dimerization. In case of PleD, response regulator receiver domains (Rec1 and Rec2) are used for this process. Many DGCs are additionally regulated by allosteric product inhibition. The above mentioned crystal structure of PleD revealed binding of intercalated c-di-GMP dimers between a primary inhibition site (I-site), represented mainly by the conserved RxxD motif (GGDEF domain), and arginines of the secondary I-site (Rec2 domain). Two contradicting modes of action were proposed for this regulatory mechanism. (I) Inhibition by c-di-GMP binding to the RxxD motif inducing conformational changes in the active site. (II) Inhibition by c-di-GMP forming cross-links between the GGDEF and the Rec2 domains and preventing hereby the formation of the dimeric active site. In this study structural, biophysical and biochemical analysis of several DGCs from C. crescentus was undertaken, to elucidate the details of the regulatory mechanisms of this class of enzymes. Analysis of the so-called ‘stand-alone’ DGCs, which consist of 25-500 amino acids long segments in front of their GGDEF domains, has shown the inability of the GGDEF domains to form dimers autonomously. The ‘stand-alone’ DGCs utilize their N-terminal segments for dimerization.
To get insights in the environmental cues dependent regulatory mechanism of dimerization, the Rec domains bearing DGC PleD was crystallized in its pseudophosphorylated/activated form. The crystal structure of BeF3!•Mg2+ activated PleD is the first structure showing a full-length response regulator in its activated state. Comparison with the structure of non-activated PleD resulted in the elucidation of the molecular mechanisms of the dimerization process. Additionally, the formation of a two-fold symmetric, charged pocket at the (Rec1-Rec2)2 stem interface was observed, which might represent the long-sought ‘pole-localization’-signal for PleD. Besides giving insights in the substrate binding mode of the DGCs, the obtained structures shed light on the catalytic mechanism of DGCs. In combination with biochemical data the structures verified the ‘two-metal assisted’ catalysis mechanism for the DGCs. A new, c-di-GMP dimer dependent domain-cross-linking mode was revealed. It is generally applicable to DGCs, involving in the process merely the GGDEF domains. It turned out that the successful inhibition of the DGC PleD relies on the presence of primary- and secondary I-sites, whereas the initial binding of c-di-GMP depends solely on the primary I-site. PleD was shown to need R390 besides the RxxD motif (the whole I-site) to bind c-di-GMP. Finally, cross-linking of proteins by c-di-GMP, intradimeric like in PleD and intermolecularly like in DgcB, was shown and might represent the main regulatory function of this second messenger.