Determinants for the Activation and Autoinhibition of the Diguanylate Cyclase Response Regulator WspR

Abstract

The bacterial second messenger c-di-GMP controls secretion, cell adhesion and motility leading to biofilm formation and increased cytotoxicity. Diguanylate cyclases containing GGDEF and phosphodiesterases containing EAL or HD-GYP domains have been identified as the enzymes controlling cellular c-di-GMP levels, yet less is known regarding the molecular mechanisms governing regulation and signaling specificity. We recently determined a product-inhibition pathway for the diguanylate cyclase response regulator WspR from Pseudomonas, a potent molecular switch that controls biofilm formation. In WspR, catalytic activity is modulated by a helical stalk motif that connects its phospho-receiver (REC) and GGDEF domains. The stalks facilitate the formation of distinct oligomeric states that contribute to both activation and autoinhibition. Here, we provide novel insights into the regulation of diguanylate cyclase activity in WspR based on the crystal structures of full-length WspR, the isolated GGDEF domain, and an artificially dimerized catalytic domain. The structures highlight that inhibition is achieved by restricting the mobility of rigid GGDEF domains, mediated by c-di-GMP binding to an inhibitory site at the GGDEF domain. Kinetic measurements and biochemical characterization corroborate a model in which the activation of WspR requires the formation of a tetrameric species. Tetramerization occurs spontaneously at high protein concentration or upon addition of the phosphomimetic compound beryllium fluoride. Our analyses elucidate common and WspR-specific mechanisms for the fine-tuning of diguanylate cyclase activity.

Cyclic di-GMP: the First 25 Years of a Universal Bacterial Second Messenger

“…However, DGC activation by secondary mechanisms derived from primary signals, e.g., protein phosphorylation, has been revealed using biochemical and structural biology approaches. Complex domain and protein subunit rearrangements that bring the GGDEF domains in close proximity have been observed by comparing X-ray structures of the (pseudo)phosphorylated and nonphosphorylated states of PleD and Pseudomonas aeruginosa WspR (PA3702; REC-GGDEF domain architecture) (86,92). Phosphorylation is a common (Table 3) and powerful mechanism for GGDEF domain activation.…”

“…5A). Additional residues coordinating binding of the c-di- GMP dimer to the I site come either from the regulatory domain, as in PleD (86), or from the GGDEF domain of another protein monomer, as in WspR or PleD (92). This allows the intercalated c-di-GMP dimer to block the GGDEF domain movement required for formation of the catalytically competent homodimer.…”

Integration of Cyclic di-GMP and Quorum Sensing in the Control of vpsT and aphA in Vibrio cholerae

“…The VpsR and YcgR proteins were purified using a 6ϫ histidine tag with affinity purification and the Impact protein purification kit from NEB, respectively, according to the manufacturer's instructions. 32 P-labeled c-di-GMP was generated using the purified cytoplasmic portion of GGDEF VC2370, which does not contain the first 142 amino acid residues (VC2370Ϫ142) (11). Reaction mixtures with a total volume of 100 l containing 10 M VC2370 in buffer (75 mM Tris-Cl [pH 7.8], 250 mM NaCl, 25 mM KCl, 10 mM MgCl 2 ) with 12.5 M [␣-32 P]GTP (800 Ci/mmol; Perkin-Elmer) or 12.5 M unlabeled GTP were incubated at room temperature for 30 min.…”

Identification of Anti-Inflammatory and Anti-Hypertensive Drugs as Inhibitors of Bacterial Diguanylate Cyclases

“…ESI-MS is particularly useful for discovery and characterization of lead compounds since it is highly sensitive and directly probe weak non-covalent protein-ligand interactions. 44 By using a highly stable isolated GG(D/E)EF domain construct of WspR (WspR GG(D/E)EF ), 15 the K d measured for sulfasalazine, folic acid, eprosartan, novobiocin, iodipamide, sulfadiazine and sulfathiazole ranged from 37 to 493 µM (Table 1 and Figure 5). Remarkably, GTP displayed a K d compatible with previously reported WspR Michaelis-Menten constant, Although the biochemical assays we performed are limited to compounds covering a reduced number of functional groups, we observed an interesting structureactivity relationship.…”

“…298 which involves c-di-GMP binding to a secondary site (I-site) characterized by a RxxD motif. [13][14][15][16] Several major human pathogens, including Pseudomonas aeruginosa, Salmonella typhimurium, and Vibrio cholerae, possess numerous c-di-GMP-metabolizing proteins, and some studies confirmed the correlation of c-di-GMP-mediated signaling with biofilm formation. [17][18][19] Furthermore, modulation of c-di-GMP signaling by environmental changes has also been linked with the dispersion of cells from established biofilms.…”