ABS 029: Comparative Molecular Dynamics Analysis of Wild-Type and Mutant Ornithine Transcarbamylase: D175G and Y176C Pathogenesis

Anna Casarino ¹, Mary Jo Ondrechen ¹, Darrin Bath ¹, Sam Shroff ¹

¹ Northeastern University

The Van Wickle Journal (2026) Volume 2, ABS029

Introduction: Ornithine transcarbamylase (OTC) is an obligate homotrimer of the transferase family that catalyzes one step in the urea cycle of mammals to sequester ammonia and synthesize citrulline. Mutations in the OTC protein in humans can cause ornithine transcarbamylase deficiency (OTCD), an X-linked urea cycle disorder characterized by the impaired ability to convert ammonia to urea, which presents as hyperammonemia. The two OTC variants investigated here, D175G and Y176C, demonstrate only mildly reduced catalytic activity and yet they cause disease. Molecular dynamics simulations are performed on these two variants of OTC, to determine whether their pathogenesis is linked to structural and dynamic changes in the protein. Resolving these mechanisms will contribute to building a general framework for predicting and understanding disease-causing mutations in OTC. Supported by NSF MCB-2147498.

Methods: Wildtype human OTC and two pathogenic variants (D175G, Y176C) were modeled from PDB structure 1C9Y. Mutations were introduced in silico using YASARA, with three carbamoyl phosphate and three ornithine ligands occupying each active site of the homotrimer. Systems were parameterized using the ff19SB force field with the OPC water model, and ligands were parameterized with GAFF2 using tleap. Each system underwent energy minimization, gradual heating, and 1 µs unrestrained production MD using pmemd.cuda (AMBER). Backbone RMSD and per-residue RMSF were calculated using cpptraj. Residue interaction networks were analyzed using RING. Principal component analysis was performed on combined trajectories to assess conformational subspace overlap across variants.

Results: All three OTC variants maintained stable backbone RMSD trajectories and comparable per-residue flexibility profiles over 1 µs. Radius of gyration analysis revealed C303 as consistently destabilized in both mutants relative to wildtype, while R92 showed reduced mobility in Y176C. PCA subspace overlap scores of 0.42–0.46 between wildtype and each mutant indicated largely non-overlapping conformational subspaces. Interaction network analysis revealed complete loss of D175/G175–R330 contact in both mutants, and selective loss of C176–S207 contact in Y176C.

Discussion: D175G and Y176C do not globally destabilize OTC, consistent with their mild catalytic impairment observed experimentally. However, PCA demonstrates that both mutations shift the conformational behavior of the broader protein, suggesting allosteric rather than direct active site disruption. Loss of D175–R330 contact in both variants may perturb the electrostatic network that positions R330 to reduce the pKa of ornithine's side chain amine, impairing nucleophilic attack on carbamoyl phosphate. The additional loss of C176–S207 in Y176C implies a distinct and potentially more disruptive interaction network perturbation, which warrants further investigation.