Identification of deleterious missense variants of serine peptidase inhibitor Kazal type 2 gene and their impact on KAZAL domain structure, stability, flexibility, and dimension

The SPINK2 protein, encoded by the SPINK2 gene, plays an essential role in the normal development of spermatozoa, and its deficiency is associated with spermatogenesis disorders ranging from aspermia to azoospermia. This study aimed to identify the most deleterious variants of the SPINK2 gene and to evaluate their effects on protein structure and function through an in silico approach. A total of 8,028 variants were identified, including 72 missense variants. Using 11 bioinformatics tools, six variants (P50L, T58I, C66Y, E62A, P42S, and P45L) were predicted to have deleterious effects. Protein-protein interaction analysis using the STRING database revealed strong functional associations between SPINK2, SPINK1, and ACR, and medium-confidence associations with SPINK4, SPINK13, PMPCA, KLK4, SPINK9, SPINK6, SPACA1, and NUDT8. Local structural analysis showed that variants such as T58I and C66Y gained additional hydrophobic interactions, whereas P50L and P42S lost key interactions, potentially impairing protein stability and function. Molecular dynamics simulations using GROMACS revealed that P50L enhances protein stability, reduces amino acid flexibility, and increases the overall dimensions of the protein. T58I had a mild effect on stability, whereas E62A and C66Y decreased stability and flexibility while increasing protein size. P42S and P45L induced slight stability alterations, reduced flexibility, and enlarged the protein. Overall, these structural and dynamic changes suggest functional impairment of SPINK2. To our knowledge, this is the first study to identify six deleterious SPINK2 variants with potential roles in the disruption of spermatogenesis, providing a foundation for future functional and clinical investigations.