P1551: conformational study of pyruvate kinase deficient variants with hemolytic anemia. the relationships between the loss of enzyme function and structural disruption

Background: Hereditary pyruvate kinase (PK) deficiency is a known cause of rare anemia characterized by accelerated destruction of red blood cells in the spleen or hemolysis. This enzymopathy shows a high clinical and genetic heterogeneity; thus, its diagnosis is sometimes difficult. The administration of an allosteric PK activator (Mitapivat) that stabilizes the enzyme and increases its enzymatic activity is recently being tested. This has led to a renewed interest in the study of the genotype-phenotype relationship in patients with PK deficiency. The assessment of the impact of a given pathogenic mutation on the conformational structure and dynamics (and thus the enzymatic activity) of the protein can enable a prediction of the possible response to treatment with the allosteric activator Mitapivat Aims: The objective of our study is to analyze the effect of the genetic mutation on the 3D structure of the enzyme and to investigate the impact on its activity and function with the insight provided by conformational modeling techniques Methods: 15 patients, homozygous or double heterozygous for PK deficiency have been included in this study. The promoter region, the exomic regions and the flanking intronic regions of the PKLR gene have been sequenced. The functional impact of the 12 identified missense mutations has been studied on the basis of the 3D structure of the enzyme. The human PK protein in the R-state was used as the starting point of the study (Protein Data Bank entry “2VGB”). Each amino acid replacement was computationally modeled. Molecular dynamics (MD) simulations were then performed on each molecular system (without ligands). The local disturbances of the mutation spots in comparison with those of the native protein during the simulations were examined as follows. Firstly, by describing the major differences in interactions between the amino acids in the vicinity of the mutation spot. Secondly, by comparing their dynamical flexibility profile in terms of the root-mean-square fluctuation metric Results: The majority of the analyzed variants display altered flexibility features in comparison with the native enzyme. A missense mutation in a given position alters the local network of amino acidic contacts. The type of functional impact of each mutation depends on the protein region where the replacement occurs Image:Summary/Conclusion: Clinical studies and structural bioinformatics should work together towards precision medicine. This joint effort will enable more accurate treatments, early diagnosis and even genetic counselling. The study of PK deficiency using computational modeling helps confirm the disruptive mechanism of pathogenic variants and predict whether the administration of an allosteric activator like Mitapivat could boost enzymatic activity. For instance, variants that affect positions involved in the binding of ligands would likely not be responsive to such treatment