Journal: Structure(London, England:1993)

Article Title: Structure of the LdcB LD-Carboxypeptidase Reveals the Molecular Basis of Peptidoglycan Recognition

doi: 10.1016/j.str.2014.04.015

Figure Lengend Snippet: Activity of Sp LdcB and Bs LdcB against Peptidoglycan and Peptides (A) HPLC chromatograms of muropeptides from S. pneumoniae strains, obtained after incubating PG with or without Sp LdcB or Sp LdcB(E204A) in the presence of Zn 2+ or EDTA, followed by digestion with cellosyl and reduction with sodium borohydride. The muropeptides are: Tri, Glc N Ac—Mur N Ac(r)—L-Ala—D-γ-Gln—L-Lys; Tetra, Glc N Ac—Mur N Ac(r)—L-Ala—D-γ-Gln—L-Lys-D-Ala; TetraTri, Glc N Ac—Mur N Ac—L-Ala—D-γ-Gln—L-Lys—D-Ala—L-Lys—D-γ-Gln—L-Ala—Mur N Ac(r)—Glc N Ac; TetraTetra, Glc N Ac—Mur N Ac—L-Ala—D-γ-Gln—L-Lys—(D-Ala)—D-Ala—L-Lys—D-γ-Gln—L-Ala—Mur N Ac(r)—Glc N Ac; deAc, deacetylation at Glc N Ac; SA, L-Ser—L-Ala branch (at L-Lys). Glc N Ac, N -acetylglusosamine; Mur N Ac(r), N -acetylmuramitol. (B) HPLC chromatograms of muropeptides from B. subtilis strains, obtained after incubating PG with or without Bs LdcB, followed by digestion with cellosyl and reduction with sodium borohydride. Muropeptides: Tri, Glc N Ac—Mur N Ac(r)—L-Ala—D-γ-Glu— meso -Dap(NH 2 ); Tetra, Glc N Ac—Mur N Ac(r)—L-Ala—D-γ-Glu— meso -Dap(NH 2 )—D-Ala; TetraTri, Glc N Ac—Mur N Ac—L-Ala—D-γ-Glu— meso -Dap(NH 2 )—D-Ala- meso -Dap(NH 2 )— D-γ-Glu—L-Ala—Mur N Ac(r)—Glc N Ac; TetraTetra, Glc N Ac—Mur N Ac—L-Ala—D-γ-Glu— meso -Dap(NH 2 )—(D-Ala)—D-Ala— meso -Dap(NH 2 )— D-γ-Glu—L-Ala—Mur N Ac(r)—Glc N Ac; meso -Dap(NH 2 ), meso -diaminopimelic acid (amidated). (C) TLC of Sp LdcB incubated with various substrates.

Article Snippet: Tetrapeptides Ala-D-Gln-L-Lys-D-Ala (Tetra-D; Activotec) or Ala-D-Gln-L-Lys-L-Ala (Tetra-L; Activotec) at 20 mM were incubated with 700 uM of zinc-loaded Sp LdcB or Sp LdcB-E153A for 2 hr at 37°C in a buffer of 10 mM HEPES/NaOH, pH 8.0, 100 mM NaCl in a reaction volume of 10 μl.

Techniques: Activity Assay, Incubation

Journal: Structure(London, England:1993)

Article Title: Structure of the LdcB LD-Carboxypeptidase Reveals the Molecular Basis of Peptidoglycan Recognition

doi: 10.1016/j.str.2014.04.015

Figure Lengend Snippet: Sp LdcB with Bound Mur N Ac—L-Ala—D-γ-Gln—L-Lys—(D-Asn) (A) Cartoon representation of Sp LdcB, drawn using the same color scheme as in Figure 3 . The Mur N Ac—Ala—D-γ-Gln—L-Lys—(D-Asn) is drawn as a ball-and-stick model with Refmac-weighted 2F obs -F calc electron density displayed at a contour level of 1 σ. (B–E) The interactions made between the ligand and the protein at each subsite are shown successive panels: (B) S 3 ; (C) S 2 ; (D) S 1 , and (E) S 1 ′. Key van der Waals’ interactions are shown as a transparent silver line. (F) A schematic of the interactions of Sp LdcB with the bound peptidoglycan mimic. Hydrogen-bond interactions are shown with a black dotted line, and van der Waals’ forces are shown with a green arc. (G) A schematic of the tetrapeptide substrate of Sp LdcB, drawn in the same manner as the peptidoglycan mimic in (F). The scissile bond is highlighted by an arrow. Crosslinks to other stem peptides would take place via the lysine’s terminal amino group on the left.

Article Snippet: Tetrapeptides Ala-D-Gln-L-Lys-D-Ala (Tetra-D; Activotec) or Ala-D-Gln-L-Lys-L-Ala (Tetra-L; Activotec) at 20 mM were incubated with 700 uM of zinc-loaded Sp LdcB or Sp LdcB-E153A for 2 hr at 37°C in a buffer of 10 mM HEPES/NaOH, pH 8.0, 100 mM NaCl in a reaction volume of 10 μl.

Techniques:

Journal: Structure(London, England:1993)

Article Title: Structure of the LdcB LD-Carboxypeptidase Reveals the Molecular Basis of Peptidoglycan Recognition

doi: 10.1016/j.str.2014.04.015

Figure Lengend Snippet: A Comparison of the Ligand-Bound Active Sites Carboxypeptidases (A) The active site of Sp LdcB (white) with bound Mur N Ac—L-Ala-D-γ-Gln—L-Lys—(D-Asn) (turquoise carbons) is compared to that of VanXYg (blue) containing a phosphinate transition state analog (pink carbons) of D-Ala—D-Ala (PDB ID 4muq ). The phosphinate in VanXYg coordinates the active site zinc in a similar manner to that of the lysyl carboxylate in the Mur N Ac—L-Ala-D-γ-Gln—L-Lys—(D-Asn) bound to Sp LdcB. The D-alanyl moiety of the phosphinate ligand in VanXYg, which occupies subsite S 1 ′, is matched by the D-alanine (green carbons) in Sp LdcB (white) and the D-Asn of the Mur N Ac—L-Ala-D-γ-Gln—L-Lys—(D-Asn) ligand. The zinc ions are shown as gray spheres. (B) The proposed positions of the two posthydrolysis ligands (orange carbons) in the active site of Sp LdcB (white). The D-alanine from the Sp LdcB structure occupies the S 1 ′ site of the protein, while the lysine side chain has been rotated and modeled in an extended conformation. The altered conformation of the lysine presents the N ζ to the surface of the protein, in an ideal position to accommodate peptide crosslinks. The zinc ions are shown as gray spheres. (C) The active sites of Sp LdcB and pancreatic carboxypeptidase A (PDB ID 1hdu [ Cho et al., 2002 ]) are compared. In each structure, the Zn 2+ ion is coordinated by two histidines (His69 and His196 in carboxypeptidase A; His153 and His207 in Sp LdcB) and an acidic amino acid (Glu72 in carboxypeptidase A; Asp160 in Sp LdcB). The reversed locations of Arg127 and Glu270 in carboxypeptidase A relative to Arg120 and Glu204 in Sp LdcB is matched by a reversal of the path of the respective substrates through the active sites and the likely retention of a common catalytic mechanism.

Article Snippet: Tetrapeptides Ala-D-Gln-L-Lys-D-Ala (Tetra-D; Activotec) or Ala-D-Gln-L-Lys-L-Ala (Tetra-L; Activotec) at 20 mM were incubated with 700 uM of zinc-loaded Sp LdcB or Sp LdcB-E153A for 2 hr at 37°C in a buffer of 10 mM HEPES/NaOH, pH 8.0, 100 mM NaCl in a reaction volume of 10 μl.

Techniques:

Journal: Structure(London, England:1993)

Article Title: Structure of the LdcB LD-Carboxypeptidase Reveals the Molecular Basis of Peptidoglycan Recognition

doi: 10.1016/j.str.2014.04.015

Figure Lengend Snippet: Conformational Changes in Sp LdcB on Ligand Binding (A and B) Cartoon representations of (A) apo Sp LdcB and (B) Sp LdcB with bound Mur N Ac-Ala-D-γ-Gln-Lys-(D-Asn) using the same color scheme as in Figure 3 . Key residues that move on ligand binding are shown in stick representation.

Article Snippet: Tetrapeptides Ala-D-Gln-L-Lys-D-Ala (Tetra-D; Activotec) or Ala-D-Gln-L-Lys-L-Ala (Tetra-L; Activotec) at 20 mM were incubated with 700 uM of zinc-loaded Sp LdcB or Sp LdcB-E153A for 2 hr at 37°C in a buffer of 10 mM HEPES/NaOH, pH 8.0, 100 mM NaCl in a reaction volume of 10 μl.

Techniques: Ligand Binding Assay