Structure of Cpe0147 with 11 repeat domains (green) and an adhesin domain (blue). Place and size of C1 and C2 constructs are indicated. (B) Two-domain C2 structure. Strands A and G are linked by an internal ester bond (yellow sticks) such that an extended covalent connectivity (indicated in blue) is propagated along the 11 repeat domains in the cell wall for the adhesin domain. Strand G is interrupted by a loop (cyan), which is shown in detail in F. Calcium ions are shown as light blue spheres. (C) Topology with the repeat domain highlights the IgG-like fold, the place of the ester bond (thick black line), plus the loop interrupting strand G. (D) Calcium site in the linker region. The Ca2+ ion is shown as a light blue sphere, the coordinated water is shown as a small red sphere, and metal igand bonds are shown as dashed yellow lines. (E) Second calcium internet site (inside repeat domain) shows two coordinated waters. (F) Close-up view with the internal ester bond in between Thr-11 and Gln-141 side chains. Side chains involved in ester bond formation or stabilization are labeled. The loop-interrupting strand G is highlighted in cyan and consists of two in the amino acids crucial to ester bond formation.1368 | pnas.org/cgi/doi/10.1073/pnas.Kwon et al.and purified in E. coli. For simplicity, the single- and two-domain constructs are referred to herein as C1 and C2, respectively. Domain boundaries of C2 were determined by restricted proteolysis, revealing a extremely stable trypsin-resistant fragment of 32 kDa (a loss of 6 kDa). Crystals of proteolyzed selenomethionine (SeMet)-substituted C2 protein were subsequently grown, along with the structure was solved by single-wavelength anomalous dispersion (SAD). The crystal structure has 1 C2 molecule per asymmetric unit and was refined at a resolution of 1.90 ?(R = 19.1 , Rfree = 21.9 ). Data collection and refinement statistics are shown in Table S1.Stalk Domain Structure. C2 is definitely an elongated molecule that is certainly 104 ?extended and 17 ?wide (Fig. 1B). It includes two IgG-like domains connected by a lengthy linker. The two repeat IgG-like domains are practically identical with an rmsd of 0.59 ?over 143 aligned C atoms. The protein forms a -sandwich consisting of opposing three- and four-stranded -sheets, comparable to an IgG continuous domain (IgGC). The C2 domain fold differs from the IgGC fold, on the other hand, in that 1 edge strand (D) is switched in the first sheet with the -sandwich to the second; the two -sheets possess the strand order A-B-E and G-F-C-D (Fig.2-Methyl-4-(trifluoromethyl)aniline web 1C).240401-09-6 Chemscene This corresponds to the switched-type Ig fold, as defined by Bork et al.PMID:28739548 (17). The interdomain linker is 35 ?lengthy comprising residues 140?52. The length of your linker implies interdomain flexibility and may perhaps allow significant domain motions. The linker is predominantly stabilized by an extended loop, residues 256?74, that projects 20 ?up from the C-terminal domain in between -strands F and G. This F-G loop stacks against the linker peptide and tends to make mostly hydrophobic interactions. A metal ion is bound in the end on the extended loop, octahedrally coordinated by Asp-267 (O1), Asp-269 (O1), Asp-271 (O1), Asn-273 (O), Asp-275 (O2), and also a water molecule (Fig. 1D). The coordination environment and average metal igand bond length (two.three ? are indicative of a Ca2+ ion, and though the C2 crystals have been grown in the presence of both 30 mM Mg2+ and 30 mM Ca2+, the metal ions are modeled as calcium (18). A second Ca2+ ion is coordinated by Asp-165 (O1) from -strand A, Asn-181 (O), Asp184 (O1), a.