C (Figure S5), although nevertheless able to bind GTP with micro-molar affinity, is totally inactive (Figure 4C and 4D), indicating that the HAMP HDAC Inhibitor site domain is vital for transient dimerization and catalysis to occur. Alternatively, the activity of YfiNHAMP-GGDEF confirms that YfiN does not undergo product feedback inhibition, no less than in vitro and in the micromolar variety that we explored (as much as 50 c-di-GMP). Likewise, Wood and coworkers have shown that in vitro feedback inhibition for fulllength YfiN is observed only at c-di-GMP concentration larger than 200 M [18]. Therefore, the YfiBNR signaling program seems to become an ON/OFF switch, with the output of the module (i.e. c-di-GMP production) responding only to external strain signals and not to endogenous c-di-GMP levels. It as been shown that the domain architecture of YfiN represents a widespread module to connect periplasmic stimuli to a cytosolic response or viceValues in parentheses refer to highest-resolution shell.GMP)two towards the I-site for sterical reasons, is observed only inside the structure of XCC4471 that also displays a degenerated I-site [31]. These evidences recommend that YfiN isn’t capable to undergo canonical solution inhibition of DGCs, implying homodimer formation in between the two catalytic domains. However, because the RxxD motif is conserved, the enzyme could still bind dimeric c-di-GMP and show item inhibition through an eventual cross-link from the GGDEF and HAMP domain, with all the second arginine provided by the latter. To verify this possibility we measured the binding affinity of YfiNHAMP-GGDEF for c-di-GMP.YfiNHAMP-GGDEF does not bind c-di-GMPBinding of c-di-GMP to YfiNHAMP-GGDEF was directly measured working with isothermal titration calorimetry (ITC) and no binding was observed (Figure 4A). Obviously an eventual misfolding with the soluble truncated construct could bias this result. To exclude this possibility we also measured the binding affinity of YfiNHAMP-GGDEF for the substrate. Binding of GTP was carried out inside the presence of CaCl2, which does not let hydrolysis right after substrate binding. YfiNHAMP-GGDEF binds GTP with submicromolar affinity and a stoichiometry close to 1 (Figure 4B). AsPLOS 1 | plosone.orgGGDEF Domain Structure of YfiN from P. aeruginosaFigure 2. Cristal structure of YfiNGGDEF. A) Cartoon representation on the YfiNGGDEF structure. The active web page and primary inhibitory website (Ip) signature residues (GGDEF and RxxD) are shown in green and magenta respectively. B) Sequence alignment in the GGDEF domain of YfiN with all the other DGCs of identified structure; PleD from C. crescentus [27,28]; WspR from P. D1 Receptor Antagonist Source aeruginosa [29]; A1U3W3 from M. aquaeolei [32] and XCC4471 from X. campestris [31]. C) Structure superposition of YfiNGGDEF together with the other DGC. YfiNGGDEF (black); PleD from C. crescentus [27,28] (grey – PDB: 2wb4 rmsd: 1.23 ; WspR from P. aeruginosa [29] (cyan PDB: 3i5a – rmsd: 1.31 ; XCC4471 from X. campestris [31] (light purple – PDB: 3qyy – rmsd: 1.64 and A1U3W3 from M. aquaeolei [32] (dark purple – PDB: 3ign – rmsd: 1.34 .doi: 10.1371/journal.pone.0081324.gPLOS 1 | plosone.orgGGDEF Domain Structure of YfiN from P. aeruginosaFigure 3. YfiN displays a degenerated Is-Site. A) Binding mode of dimeric c-di-GMP to the I-site of DGCs or to receptor proteins. The very first row shows the homo-domain cross-linking (GGDEF/GGDEF), when the second shows the hetero-domain cross-linking (inside the same chain) of inhibited PleD and two c-di-GMP receptors. For all structures diff.