STRUCTURAL CHARACTERIZATION OF A DIGUANYLATE CYCLASE FROM THERMOTOGA MARITIMA

Abstract

Cyclic-di-GMP is a second messenger in bacteria and is involved in signal transduction pathways. This molecule has been shown to bind to the GGDEF, EAL and HD-GYP domain containing proteins, regulating a variety of bacterial functions. Diguanylate cyclases (DGCs) have the GGDEF domain that catalyzes the formation of cyclic-di-GMP from two GTP molecules. Experimental studies of cyclic-di-GMP pathways and development of anti-virulence agents necessitate milligram quantities of c-di-GMP. Since chemical synthesis of cyclic-di-GMP is time-consuming and expensive, an enzymatic approach, using enzymes such as PleD and WspR, is being used to produce cyclic-di-GMP on a laboratory scale. However, these enzymes yield significantly low amounts of cyclic-di-GMP due to poor thermostability and strong product inhibition. Furthermore, the produced cyclic-di-GMP mediates feedback inhibition by binding to a conserved motif , called I site, in the enzyme. The Liang lab of NTU university have reported a stand-alone GGDEF domain from Thermotoga maritima with a mutation at the inhibitory site (R158A) which is devoid of any feedback inhibition and synthesizes milligram quantities of cyclic-di-GMP. This mutant also exhibits a good half-life due to improved thermostability. Although recently mesophilic enzymes with mutation at the I site have been used, the use of a thermostable enzyme is advantageous as it remains active at room temperature for several days enabling continuous synthesis. This current study involves the structural characterization of the standalone GGDEF domain DGC from this thermophile. The GGDEF domain has been crystallized in an inhibited conformation with cyclic-di-GMP bound at the inhibitory site, mediating dimer formation. The bridging of the two protein chains by cyclic-di-GMP in this structure, at 2.27 Å, is indicative of inhibition by domain immobilization, where the active sites are pushed apart forming an inactive conformation as observed in homologous structures. Also, the structure of the apo form of the I site mutant (R158A) has been solved at 2.5 Å resolution. A third crystal form in an active-like dimeric conformation with the active sited in proximity was used to develop a model of the catalytically active dimer. Comparison of the GGDEF domains with and without the ligand, suggests that the overall structure is similar and binding of dimeric ligand at the I site creates no changes at the active site indicating an absence of intrinsic allostery. Additionally, a comparison of the electrostatic interactions, hydrogen bonds and other parameters known to contribute to thermostability indicated that the higher thermostability of this enzyme is solely due to the large network of ion pairs. This was also verified by thermal denaturation experiments, which suggested that salt bridge forming residues at loop regions contribute more to thermostability than those connecting secondary structures.