Fig. 5: Recognition of collagen acceptor peptide and catalytic mechanism of GLT25D1.
From: Molecular basis of collagen galactosylation by GLT25D1
A Sequence conservation of the 14 confirmed galactosylation sites in human collagen type II (generated by WebLogo). The logo size is proportional to the level of conservation. The hydroxylysine (Hyl) and its following glycine residues are absolutely conserved. B Close-up view of the CGT active site in the ternary complex structure of GLT25D1 (in gray surface rendering) with collagen acceptor peptide (yellow stick) and UDP (cyan stick). The acceptor peptide is positioned within a binding cleft, adjacent to UDP. GLT25D1 residues lining the acceptor-binding interface are shown as blue sticks. Yellow dashed lines indicate potential hydrogen bond or salt bridge. C The acceptor peptide (yellow) and its surrounding GLT25D1 residues (blue) are superimposed with their corresponding densities (gray mesh; contour level 0.022). D Side view of the acceptor peptide binding cleft, rotated by 45 degrees relative to that in (B). Pink dashed lines indicate distances between groups as labeled. E Relative activity of wild-type (WT) GLT25D1 and mutants targeting residues in the acceptor binding site as well as catalytic residue D522 as identified in (C). Activities were normalized to the amount of protein. Data are presented as mean ± s.d. for three independent replicates. Statistical significance was determined using ordinary one-way ANOVA, multiple comparison test (Dunnett’s test at 95% CI) and differences are depicted as **p < 0.01; ****p < 0.0001; ns not significant. The exact p-values are as follows. For the peptide with hydroxylysine substrate: WT vs Control (p < 0.0001), K470A (p < 0.0001), Y494A (p < 0.0001), W495A (p < 0.0001), Y561A (p < 0.0001), T562A (p < 0.0001), D522A (p < 0.0001) and E523A (p < 0.0001). For the Gelatin substrate: WT vs Control (p < 0.0001), K470A (p < 0.0001), Y494A (p < 0.0001), W495A (p < 0.0001), Y561A (p = 0.9997), T562A (p < 0.0001), D522A (p < 0.0001) and E523A (p = 0.0037). Source data are provided as a Source Data file. F Proposed SN2 single-displacement reaction mechanism of GLT25D1. Specifically, D522 deprotonates the 5-hydroxyl group of hydroxylysine, activating it as a nucleophile to attack the anomeric carbon (C1) of the galactose moiety.
