Fig. 3: A two-state induced fit model of cooperative UDP-glucose binding.

a Ribbon diagram of the R699 active site bound to Mn²⁺ (royal purple) and UDP (yellow). The uridine ring of UDP is sandwiched between W48 and Y85. Additional key active site residues are labeled. b Structural comparison of R699 with and without UDP bound, shown as cartoon diagrams. The UDP-bound structure is in purple, and the apo form is in cyan. Key active site residues undergoing conformational changes are highlighted, with UDP shown in yellow. c Enzymatic activity assay of R699 using galactosylhydroxylysine as the substrate. Enzymatic activity was measured by detecting UDP production with a luciferase assay. Results are shown as mean values (± S.D.) from triplicate biological samples (n = 3). p values were determined using two-tailed Student’s t-tests. Although p values between wild-type (WT) and mutant proteins are not displayed, they are <0.001. d Sequence alignment of R699 with human PLOD2, highlighting K438 in R699 and its corresponding lysine residue in PLOD2 in bold. e Ribbon diagram of the K222-containing helix and the dimer-stabilizing loop (highlighted within a gray oval) in R699. Structures with UDP bound (purple) and without UDP (cyan) are overlaid. Residues undergoing conformational changes are shown as sticks and labeled. f Schematic model illustrating the proposed mechanism of positive cooperative interactions between the two active sites within the R699 dimer during UDP-glucose binding. GLT and AC domains are in purple and orange, respectively.