**Background:** Pyruvate kinase deficiency (PKD) is a rare autosomal recessive disorder caused by mutations in the PKLR gene, leading to defective pyruvate kinase enzyme activity, reduced ATP production in erythrocytes, and subsequent hemolytic anemia. Clinical features include anemia, jaundice, splenomegaly, and gallstones, but diagnosis requires enzyme activity assays or genetic sequencing. Over 300 PKLR mutations have been reported, but many novel variants remain uncharacterized. Molecular dynamics (MD) simulations can predict how mutations affect protein structure and function.
**Methods:** The proband was a 2-year-old girl with severe progressive jaundice, anemia (hemoglobin 21 g/L, RBC 1.00×10^12/L), elevated reticulocytes (13.1%), total bilirubin (43.4 μmol/L), and lactate dehydrogenase (1255.3 U/L). Whole exome sequencing identified compound heterozygous PKLR mutations: a frameshift (c.1097del, p.P366Lfs*12) inherited from the father and a missense (c.1493G>A, p.R498H) from the mother. Homology modeling used PDB ID 6NN7 to generate 3D structures of wild-type (WT) and mutant proteins. MD simulations (50 ns) were performed using GROMACS with Amber14SB force field, analyzing RMSD, RMSF, Rg, SASA, and secondary structure. Bioinformatic tools (DynaMut, Novopro, PONDR) predicted stability, hydrophobicity, and disorder. In vitro, WT and mutant PKLR constructs were overexpressed in 293T cells, and protein expression was assessed by Western blot.
**Key Results:** MD simulations showed that the WT protein had RMSD 0.216±0.032 nm, R498H 0.262±0.039 nm, and P366Lfs*12 0.713±0.094 nm, indicating reduced stability for both mutants. Rg values were WT 2.456±0.008 nm, R498H 2.501±0.019 nm (less compact), and P366Lfs*12 2.113±0.051 nm (more compact). RMSF analysis revealed increased flexibility in R498H near residues E304, G305, and V39, while P366Lfs*12 showed decreased flexibility in the overlapping region (S144-L146). SASA values were WT 239.540±3.423 nm², R498H 248.179±3.029 nm², and P366Lfs*12 169.338±6.173 nm². Secondary structure analysis showed R498H had a 1.12% reduction in α-helix content, while P366Lfs*12 had a marked decrease in α-helices. Structural superposition during the stable period (40-50 ns) revealed a loop displacement near R498H and a β-sheet-to-coil change in P366Lfs*12. Bioinformatic analysis indicated R498H increased hydrophobicity (score 0.7) and decreased intramolecular interactions and intrinsically disordered regions. Western blot showed significantly reduced expression of both mutant proteins compared to WT, with the truncated P366Lfs*12 protein not detected.
**Clinical Implications:** This study identifies a novel compound heterozygous PKLR mutation causing severe PKD, expanding the known mutation spectrum. MD simulations effectively predicted structural destabilization and loss of function, which were confirmed by reduced protein expression in vitro. These findings support the use of computational methods to interpret variant pathogenicity, especially for rare mutations. Genetic counseling is crucial for families with hereditary anemia, as the proband's sister also carried the same mutations and had α-thalassemia. Emerging therapies, such as the allosteric activator mitapivat and gene editing strategies, offer hope for future treatment, though long-term efficacy and safety require further study.