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Molecular dynamics simulations refine the pathogenicity of ACVRL1 kinase domain variants by quantifying impacts on ATP binding in pulmonary arterial hypertension
Single amino acid substitutions in the ATP-binding domain of ACVRL1, a key receptor in the bone morphogenetic protein (BMP) signaling pathway, are frequently classified as variants of uncertain significance (VUS), complicating molecular diagnosis for pulmonary arterial hypertension (PAH) and Hereditary Hemorrhagic Telangiectasia (HHT). Since aberrant ATP binding disrupts downstream SMAD1/5/8 phosphorylation, we employed molecular dynamics (MD) simulations to quantitatively assess the functional impact of these variants. We first validated our approach on 20 known pathogenic/likely pathogenic variants within 5Å of the ATP-binding site, finding that 18 (90%) caused significant alterations in binding affinity (|d| ≥ 0.8, p < 0.001). We then applied this protocol to all known VUS, conflicting, and unclassified variants within the same region, reclassifying 20 of 32 (63%) as likely pathogenic. Comprehensive in silico mutagenesis of all possible substitutions at ATP-binding pocket positions, combined with InterVar classification under HHT phenotype, enabled reclassification of 9 of 12 (75%) VUS as likely pathogenic. Finally, we demonstrated the applicability of this approach in two PAH patients with HHT carrying ACVRL1 VUS. This work establishes MD simulation of ATP-binding affinity as an effective and scalable tool for the functional interpretation of kinase variants, with broad potential for application across other disease-associated kinases.