Fig. 2
From: Molecular architecture of the multifunctional collagen lysyl hydroxylase and glycosyltransferase LH3
Insights into LH3 glycosyltransferase activity. a Co-crystallizations with Mn2+ and donor substrates revealed clear electron density (2Fo-Fc omit electron density maps, green mesh, contour level 1.2 σ) for the metal ion and for UDP in the catalytic site of the N-terminal GT domain. Residues involved in coordination of the metal ion are shown with orange sticks, while residues interacting and stabilizing UDP binding are shown in blue. Residue Asn223, found mutated into a Ser and causing pathogenic phenotypes similar to osteogenesis imperfecta, is shown with magenta sticks. b Binding of donor substrates induces conformational changes in the GT domain. Shown is a superposition of ligand-free (yellow), Mn2+-bound (blue), and Mn2+-donor substrate-bound (violet) structures of LH3 GT domain. Only the UDP-Gal-bound structure is shown; the conformation observed in UDP-Glc-bound structure is identical. Flexible loop 72–87 becomes well defined only in donor substrate-bound structures; in these structures, residues Trp145 and Trp148 adopt different conformations. c Luminescence-based assays for the evaluation of LH3 GT enzymatic activity show that mutants W75A, Y114A, and N223S are inactive. Control experiments were performed without adding enzyme. Error bars represent standard deviations from average of triplicate independent experiments. Statistical evaluations based on pair sample comparisons between uncoupled and coupled assay values using Student’s t-test. *P-value <0.05; ***P-value <0.001. d Details of the interface between the N-terminal (GT, blue) and the central (AC, orange) LH3 glycosyltransferase domain. Residues found at the interface are shown as sticks (side chain view only). The disulfide bond found in the linker region between the two domains is shown with yellow spheres on the sulfur atoms
