Pulmonary Arterial Hypertension (PAH) is a severe, disabling disease characterized by an increase in pulmonary vascular resistance against a background of normal left ventricular filling pressure. The pathogenesis of PAH is frequently associated with dysfunction of the transforming growth factor-β/ bone morphogenetic protein type 2 receptor (TGF-β/BMPR2) signaling pathway. This pathway dysfunction has a genetic foundation, as a significant proportion of patients carry loss-of-function mutations in BMPR2 gene.

Activin A receptor like type 1 (ACVRL1) is another gene belonging to the TGF-β signaling superfamily, mutations in which are associated with PAH and, in particular with Hereditary Hemorrhagic Telangiectasia (HHT) or Rendu-Osler-Weber disease. ACVRL1 encodes ALK1 (Activin receptor-like kinase 1), a type I transmembrane serine/threonine kinase receptor that is predominantly expressed on vascular endothelial cells and plays an essential role in regulating vascular remodeling and angiogenesis. Upon binding of ligands such as BMP9 and BMP10, ALK1 forms a heteromeric complex with the type II receptor BMPR2 and the co-receptor endoglin (ENG), activating downstream SMAD1/5/8 signaling and promoting endothelial quiescence and vessel maturation.

Molecular Dynamics (MD) is widely used to analyze the conformational dynamics of proteins and nucleic acids, study ligand binding mechanisms, predict the stability of mutant proteins, and model membrane systems (Diaz et al., 2022). MD can provide mechanistic insights into the pathogenicity of amino acid substitutions, especially those occurring in protein active sites.

Here we utilize MD simulation to evaluate how single amino acid substitutions in the serine-threonine kinase domain of ACVRL1 affect ATP binding. Since ATP acts as a phosphate group donor for SMAD transcription factor phosphorylation, changes in binding efficiency directly impact SMAD activation. The effect of a mutation can be quantified by calculating the binding free energy of the ACVRL1-ATP complex, which reflects the stability of ATP within the binding pocket.

Single amino acid substitutions in the ATP-binding domain of ACVRL1 are frequently classified as variants of uncertain significance (VUS) owing to insufficient support from computational predictors. A VUS classification means that there is insufficient or conflicting evidence regarding a molecular alteration's role in disease, which complicates patient counseling and clinical management. Functional data has been shown to be one of the best types of evidence for the reclassification of VUS, and the ACMG/AMP framework recognizes well-established functional studies as strong evidence under the PS3/BS3 criteria. However, such assays are rarely available for novel or private variants. This study aimed to establish MD-derived ATP-binding free energy calculations as a reproducible and scalable functional evidence criterion for the reclassification of ACVRL1 VUS, and to demonstrate its utility in patients with PAH and HHT carrying variants of uncertain clinical significance.