Investigating the Allosteric Regulation of YfiN from Pseudomonas aeruginosa: Clues from the Structure of the Catalytic Domain

Abstract

Pseudomonas aeruginosa is responsible for a plethora of biofilm mediated chronic infections among which cystic fibrosis pneumonia is the most frightening. The long-term survival strategy of P. aeruginosa in the patients lungs is based on a fine balance of virulence vs dormant states and on genetic adaptation, in order to select persistent phenotypes as the small colony variants (SCVs), which strongly correlate with antibiotic resistance and poor lung function. Recent studies have coupled SCV with increased levels of the signaling molecule cyclic di-GMP, and demonstrated the central role of the diguanylate cyclase YfiN, part of the tripartite signaling module YifBNR, in c-di-GMP dependent SCV regulation. YfiN, also called TpbB, is a multi-domain membrane enzyme connecting periplasmic stimuli to cytosolic c-di-GMP production by an allosteric inside-out signaling mechanism that, due to the lack of structural data, is still largely hypothetical. We have solved the crystal structure of the catalytic domain (GGDEF), and measured the enzymatic activity of the cytosolic portion in real-time by means of a newly developed method. Based on these results we demonstrate that, unlike other diguanylate cyclase, YfiN does not undergo product feedback inhibition, and that the presence of the HAMP domain is required for dimerization and catalysis. Coupling our structural and kinetic data with an in silico study we are now able to propose a model for the allosteric regulation of YfiN.

In Silico Discovery and In Vitro Validation of Catechol-Containing Sulfonohydrazide Compounds as Potent Inhibitors of the Diguanylate Cyclase PleD

“…Purification of the DGCs from P. aeruginosa, i.e., (i) YfiN HAMP-GGDEF (cytosolic portion containing the HAMP-GGDEF domain), (ii) YfiN GGDEF (construct containing the GGDEF domain), and (iii) WspR, was performed as previously described (14,22).…”

“…We performed the experiment with YfiN GGDEF , which is a more suit- able system for denaturation experiments than PleD or WspR, given that its oligomeric state is not nucleotide dependent (14). We found that the presence of excess compound 7 (100 M) does not affect the secondary-structure content and the overall stability of the protein, thus indicating that the observed inhibitory property of compound 7 with DGCs is not due to unspecific folding effects (see Fig.…”

“…Moreover, according to our structural model, compound 7 should target the active site of DGCs. As mentioned above, many DGCs, including PleD and WspR, are allosterically inhibited by c-di-GMP (via the I site); to exclude that compound 7 targets the I site, we tested its effect on a third DGC, i.e., the cytosolic portion of YfiN (here YfiN GGDEF-HAMP ) from P. aeruginosa, which is known to be insensitive to feedback inhibition of c-di-GMP (due to the presence of a degenerated I site) (14). The residual activity of YfiN HAMP-GGDEF in the presence of 100 M compound 7 (Fig.…”

“…In order to bypass this limitation, we designed a FRET-based displacement experiment aimed at demonstrating qualitatively that inhibitor 7 competes for the GTP binding site. The experiment was done with the DGC YfiN GGDEF , for which the binding affinity for GTP is quantitatively demonstrated (14), in complex with a stoichiometric amount of the GTP analogue MANT-GTP, which saturated the protein (see Fig. S2 in the supplemental material for details), and addition of different concentrations of inhibitor 7.…”

“…However, these compounds are likely ineffective against those DGCs lacking an I site (14) and are linearized in vitro by the EAL subtype of PDEs, if one of the natural phosphodiester bonds is conserved (15).…”