Extended Data Fig. 5: Prediction of glycerol gradient-sedimented CAD oligomers, and observation of cell cycle-independent CAD oligomerization.
From: Allosteric regulation of CAD modulates de novo pyrimidine synthesis during the cell cycle
a,b, Coomassie blue- (a) or silver-stained (b) 4–12% Bis-Tris gel, loaded with a glycerol gradient molecular standard mixture of thyroglobulin, apoferritin, β-amylase, and bovine serum albumin differentially resolved through glycerol gradient rate zonal centrifugation. The results were reproducible over 5 times ((a) and (b) are independent). c, Relative abundance of the silver-stained standards in (b). d, Estimation of glycerol gradient fractions corresponding to CAD oligomers by interpolating CAD oligomer molecular sizes into a regression line generated by the sedimentation information of standard molecules. e, A crystal structure of aspartate transcarbamoylase (ATCase) domain of CAD with R2187 residue highlighted in red (PDB: 5G1O; apo). Each monomer of the ATCase trimer is distinctly colored. f, A crystal structure of dihydroorotase (DHOase) domain of CAD with M1601 residue highlighted in red and pink (PDB: 4C6D; N-carbamoyl-L-aspartate (substrate) bound). Each monomer of the DHOase dimer is distinctly colored. g, Western blots of CAD differentially expressed in R2187A, M1601E, or R2187A/M1601E CAD mutant-expressing sgCAD single cell clones selected. The single cell clones with a red asterisk were mainly tested for CAD activity estimation in this report. h, Western blots of CAD of DTB-synchronized wild-type HeLa cell lysates differentially sedimented throughout a 10–35 % glycerol gradient by rate-zonal centrifugation (left). Quantification of CAD protein intensity from the western blotting results (right). i, Western blots of CAD of NOC-synchronized wild-type HeLa cell lysates differentially sedimented throughout a 10–35 % glycerol gradient by rate-zonal centrifugation (left). Quantification of CAD protein intensity from the western blotting results (right).
