C (Figure S5), although still in a position to bind GTP with micro-molar affinity, is entirely inactive (Figure 4C and 4D), indicating that the HAMP domain is important for transient dimerization and catalysis to occur. However, the activity of YfiNHAMP-GGDEF confirms that YfiN does not undergo item feedback inhibition, a minimum of 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]. As a result, the YfiBNR signaling technique seems to become an ON/OFF switch, with all the output from the module (i.e. c-di-GMP production) responding only to external anxiety 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 for the I-site for sterical motives, is observed only inside the structure of XCC4471 that also displays a degenerated I-site [31]. These evidences suggest that YfiN just isn’t in a position to undergo canonical product inhibition of DGCs, implying homodimer formation amongst the two catalytic domains. On the other hand, since the RxxD motif is conserved, the enzyme could nevertheless bind dimeric c-di-GMP and show product inhibition via an eventual cross-link with the GGDEF and HAMP domain, together with the second arginine offered 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 making use of isothermal titration calorimetry (ITC) and no binding was observed (Figure 4A). Certainly an eventual misfolding on the soluble truncated construct could bias this outcome. To exclude this possibility we also measured the binding affinity of YfiNHAMP-GGDEF for the substrate. Binding of GTP was carried out within the presence of CaCl2, which will not allow hydrolysis immediately after substrate binding. YfiNHAMP-GGDEF binds GTP with submicromolar affinity in addition to a stoichiometry close to one particular (Figure 4B). AsPLOS One | 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 major inhibitory website (Ip) signature residues (GGDEF and RxxD) are shown in green and magenta respectively. B) Sequence alignment from the GGDEF domain of YfiN using the other DGCs of identified structure; PleD from C. crescentus [27,28]; WspR from P. Caspase 2 Inhibitor Storage & Stability aeruginosa [29]; A1U3W3 from M. aquaeolei [32] and XCC4471 from X. campestris [31]. C) Structure superposition of YfiNGGDEF 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 JAK1 Inhibitor web 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 One | plosone.orgGGDEF Domain Structure of YfiN from P. aeruginosaFigure 3. YfiN displays a degenerated Is-Site. A) Binding mode of dimeric c-di-GMP for the I-site of DGCs or to receptor proteins. The initial row shows the homo-domain cross-linking (GGDEF/GGDEF), whilst the second shows the hetero-domain cross-linking (within the exact same chain) of inhibited PleD and two c-di-GMP receptors. For all structures diff.
