C (Figure S5), despite the fact that still capable to bind GTP with micro-molar affinity, is completely inactive (Figure 4C and 4D), indicating that the HAMP domain is essential for transient dimerization and catalysis to occur. However, the activity of YfiNHAMP-GGDEF confirms that YfiN will not undergo product CaMK II Inhibitor Synonyms feedback inhibition, at least in vitro and in the micromolar range 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 . Therefore, the YfiBNR signaling method seems to be an ON/OFF switch, using the output on the module (i.e. c-di-GMP production) responding only to external tension signals and to not 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 for the I-site for sterical motives, is observed only inside the structure of XCC4471 that also displays a degenerated I-site . These evidences recommend that YfiN will not be capable to undergo canonical product inhibition of DGCs, implying homodimer formation amongst the two IL-2 Modulator Compound catalytic domains. On the other hand, since the RxxD motif is conserved, the enzyme could nevertheless bind dimeric c-di-GMP and show solution inhibition via an eventual cross-link with the GGDEF and HAMP domain, using the second arginine supplied by the latter. To verify this possibility we measured the binding affinity of YfiNHAMP-GGDEF for c-di-GMP.YfiNHAMP-GGDEF will not bind c-di-GMPBinding of c-di-GMP to YfiNHAMP-GGDEF was directly measured using isothermal titration calorimetry (ITC) and no binding was observed (Figure 4A). Certainly an eventual misfolding of 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 will not enable hydrolysis immediately after substrate binding. YfiNHAMP-GGDEF binds GTP with submicromolar affinity along with a stoichiometry close to 1 (Figure 4B). AsPLOS A single | plosone.orgGGDEF Domain Structure of YfiN from P. aeruginosaFigure 2. Cristal structure of YfiNGGDEF. A) Cartoon representation on the YfiNGGDEF structure. The active website and principal inhibitory web page (Ip) signature residues (GGDEF and RxxD) are shown in green and magenta respectively. B) Sequence alignment from the GGDEF domain of YfiN together with the other DGCs of identified structure; PleD from C. crescentus [27,28]; WspR from P. aeruginosa ; A1U3W3 from M. aquaeolei  and XCC4471 from X. campestris . 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  (cyan PDB: 3i5a – rmsd: 1.31 ; XCC4471 from X. campestris  (light purple – PDB: 3qyy – rmsd: 1.64 and A1U3W3 from M. aquaeolei  (dark purple – PDB: 3ign – rmsd: 1.34 .doi: 10.1371/journal.pone.0081324.gPLOS One | plosone.orgGGDEF Domain Structure of YfiN from P. aeruginosaFigure three. 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), even though the second shows the hetero-domain cross-linking (within precisely the same chain) of inhibited PleD and two c-di-GMP receptors. For all structures diff.