Plosone.orgGGDEF Domain Structure of YfiN from P. aeruginosaFigure two. Cristal structure
Plosone.orgGGDEF Domain Structure of YfiN from P. aeruginosaFigure two. Cristal structure of YfiNGGDEF. A) Cartoon representation of the YfiNGGDEF structure. The active web page and major inhibitory web-site (Ip) signature residues (GGDEF and RxxD) are shown in green and magenta respectively. B) Sequence alignment from the GGDEF domain of YfiN with all the other DGCs of identified structure; PleD from C. crescentus [27,28]; WspR from P. MDH1 Protein Gene ID 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: ten.1371journal.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 towards the I-site of DGCs or to receptor Ephrin-B2/EFNB2 Protein Purity & Documentation proteins. The very first row shows the homo-domain cross-linking (GGDEFGGDEF), although the second shows the hetero-domain cross-linking (within the same chain) of inhibited PleD and two c-di-GMP receptors. For all structures various colors are applied to illustrate domains belonging to different subunits, the side chains of the two arginines and also the aspartic acid (R1; R2 and D) are shown as sticks, although the two bound c-di-GMP molecules as balls and sticks. Grey continuous lines indicate H-bonds, although green continuous lines highlight the -cation interaction among a charged nitrogen atom with the arginine residues and the guanine delocalised technique. Ip and Is indicate principal and secondary inhibitory websites respectively. Starting from top rated left, the reported structure are: PleD (PDB: 2v0n [28]); WspR (PDB: 3bre [30]); A1U3W3 (PDB: 3ign [32]); PleD (PDB: 1w25 [27]); PelD (PDB: 4dn0 ) [33]. and PP4397 (PDB: 3kyf [34]). B) Comparison from the I-site of YfiN and (PDB: 2v0n [28]). The two subunits of a hypothetical inhibited dimer of YfiN (superposed on the structure of PleD) are shown in white and pink, even though the exact same color code of panel A is employed for PleD. C-diGMP molecules (bound to PleD) are shown as lines. YfiN lacks two from the 3 arginine residues binding to c-di-GMP via the stair motif interaction (D273 and N351 – bold labels). Additionally, the presence of a bulky side chain (Y379) yields a shift of helix-A, implying a lowered, sub optimal, volume in the I-site.doi: 10.1371journal.pone.0081324.gPLOS 1 | plosone.orgGGDEF Domain Structure of YfiN from P. aeruginosaFigure 4. Binding affinity for nucleotides and enzymatic activity of YfiNHAMP-GGDEF and YfiNGGDEF. For all ITC experiments upper panels show the Raw ITC data, when lower panels show the integrated peak locations (black square) fitted using the one-bindingsite model of ORIGIN offered by MicroCal (continuous lines). Derived thermodynamic parameters are listed in Table two A) Microcalorimetric titration of 3 M YfiNHAMP-GGDEF with c-di-GMP (90 M within the syringe). No binding was observed either inside the presence of CaCl2 or inside the presence of MgCl2MnCl2 (information not shown). No thermodynamic parameters have been derived. B) Microcalorimetric titrations of 14 M enzyme answer with GTP (170 M within the syringe). The thermodynamic profile indicates that the interaction of YfiNHAMP-GGDEF with GTP presents favorable binding enthalpy and entropy, which sug.