Extended Data Fig. 9: Proposed mechanisms of UTP-mediated allosteric regulation in wild-type HeLa, G-Loop1, and T′-loop chimera CAD cells and verification of cell cycle synchronization of T′-loop chimera CAD mutant cell line.
From: Allosteric regulation of CAD modulates de novo pyrimidine synthesis during the cell cycle
a, Structural comparisons of human CAD allosteric domain structure(s) homology-modeled against E. coli carB or human CPS1. Crystal structure of E. coli carB allosteric domain (top left), two human CPS1 allosteric domains (top right; allosteric activator-bound (active state) and apo (inactive state)). Predicted human CAD allosteric domain structures homology-modeled against E. coli carB (bottom left), human CPS1 (bottom right). T′-loop colored green (active), red (inactive). Allosteric ligands shown. Annotated residues exemplify differences in structure between E. coli carB- and human CPS1-based models. b, CAD phylogenetic tree. Uniprot codes in parenthesis. Human CAD, CPS1, and E. coli carB, colored red. c, Overview of pairwise alignment. Percentage identity noted below. d, Docking simulations of UTP-relevant metabolites in human CPS1-based allosteric domain structure of human CAD. e, Alignment of human CAD CPS2 and CPS1 T′-loops. f, Relative frequencies of residue interactions between T′-loop and other domains of human CAD CPS2. Analyses based upon two structures of human CAD CPS2, predicted by homology modeling against human CPS1 structures (PDB: 5DOU, active; 5DOT, inactive). T′-loop-interacting residues in active state (top) or inactive state (bottom) model quantitated via counting neighboring residues of conserved or divergent residues of T′-loop. Human CAD T′-loop residues conserved in human CPS1 are green; divergent, red. Allosteric domain of human CAD, grey. g, Proposed mechanism of CAD allosteric regulation. When [UTP] < Kd of UTP for CAD allosteric domain, Loop1 and Loop2 interact with and stabilize T′-loop, leading to substrate channel formation; active CAD. When [UTP] > Kd, UTP binds to allosteric domain and recruits Loop1 and Loop2, destabilizing T′-loop and substrate channel; inactive CAD. In G-Loop1 mutant, Loop1 constantly interacts with T′-loop, inducing T′-loop stability and substrate channel formation; constitutively active CAD. In T′-loop chimera, [UTP] > Kd, UTP binds to the allosteric domain and recruits Loop1 and Loop2. Because of T′-Loop replacement, loop1-T’-loop interactions and stability regulation are perturbed; no UTP-mediated allosteric regulation. h,i, Cell parameter (h) and EdU incorporation (i) of T′-loop chimera, NOC synchronized. Data are the mean ± s.d. (n = 3, technical replicates, 2 independent experiments).
