Ier electron density map, 24s in comparison to 10s for the second strongest web page, which corresponds to a sulphur atom of a cysteine residue within the structure. The metal binding web page is situated on the opposite side on the plausible active web page cleft, held by the loop in the “grip” motif described above at the same time because the N- and C-terminal regions in the Cip1 core domain. The nature of this possible metal atom was unknown, therefore many atoms were modelled during the refinement. A calcium atom wasfound to provide the ideal fit with regards to both B aspect and metal coordination geometry. To further confirm the identity in the metal bound to the protein, a sample of Cip1 was characterised by particle-induced X-ray emission (PIXE). The PIXE spectrum (information not shown) unambiguously identified the presence of 1 calcium atom bound for every single Cip1 molecule in resolution.Figure five. The “grip” motif in Cip1 in comparison to glucuronan lyase from H. jecorina. The grip motif is often a conserved area in Cip1, both TrkA Inhibitor drug sequentially and structurally, right here displaying Cip1 (green) superposed towards the glucuronan lyase from H. jecorina (red). In these two structures, there’s a string of homologous residues which are positioned across the “palm” b-sheet (vibrant colours). The loop representing the “bent fingers” participates in binding a calcium ion represented as a sphere. The conserved coordinating aspartate can also be shown in Nav1.1 Inhibitor Accession bright colours. Asn156 in Cip1 binds a N-acetyl glucosamine molecule but the equivalent residue in the glucuronan lyase is actually a non-glycosylated aspartate. Numerous from the residues which might be not identical are however comparable in physical properties. doi:10.1371/journal.pone.0070562.gFigure six. The calcium binding internet site in Cip1 compared to glucuronan lyase from H. jecorina. The calcium binding internet site located in the Cip1 structure. Cip1 structure (green) superposed to the glucuronan lyase structure from H. jecorina (red). Asp206 is shown in bright colours because it truly is sequentially and structurally conserved and it coordinates the calcium ion together with the two side chain oxygen atoms (also ??see Figure 8). All coordination distances are in between 2.3 A and two.6 A. doi:ten.1371/journal.pone.0070562.gPLOS One particular | plosone.orgCrystal Structure of Cip1 from H. jecorinaFigure 7. Comparison of Cip1 to alginate lyase from Chlorella virus at pH 7 and pH 10. Superposition of Cip1 from H. jecorina (green) to the alginate lyase from Chlorella virus (blue) plus the interactions with bound D-glucuronic acid (violet) at A) pH 7 and B) pH ten. The residues are numbered in line with the Cip1 structure. Plausible catalytic residues are brightly coloured inside the figure. Water molecules are depicted in red and belong for the structure of Cip1. Panel A displays the alginate lyase structure at pH 7, the D-glucuronic acid interacts together with the glutamine in the best on the active cleft. The corresponding glutamine in Cip1 (Gln104) instead forms a hydrogen bond to a water molecule, that is also bound by Asp116, a residue which has dual conformations in Cip1. Panel B displays the alginate lyase structure at pH ten, the D-glucuronic acid interacts with Arg100 in the reduced end in the cleft. Both Asp116 and His98 in Cip1 show dual conformations pointing toward this position which may perhaps be an indication that the area is dynamic and that these residues are somehow involved in substrate binding. Asp116 and His98 don’t have any equivalents in the lyase structure. doi:ten.1371/journal.pone.0070562.gWhether calcium has any part inside the.