This study explored the structural and potential functional impact of four SPHK1 mutations, primarily P89S, A186T, and G369S, identified in prostate cancer samples. Through a combination of sequence alignment, conservation analysis, and advanced structural modeling, we assessed whether these variants could induce conformational changes or modify enzymatic activity in sphingosine kinase 1 (SPHK1), a protein known to regulate bioactive lipid signaling pathways implicated in oncogenesis.

Our results show that although none of the studied mutations disrupted the global fold of SPHK1, each introduces distinct local alterations in side-chain properties, flexibility, or packing (Table 1). The P89S mutation substitutes proline, a rigid and helix-breaking residue, for a more flexible serine. This change, occurring near a loop on the surface, may increase flexibility and modulate interactions with other proteins or membranes. A186T introduces a polar threonine side chain into the hydrophobic core, potentially altering local packing and forming new hydrogen bonds. While subtle, this change may influence the structural integrity or dynamics of the central domain. G369S replaces glycine, the most conformationally flexible residue, with serine in a lower-confidence region. This substitution likely restricts backbone motion in areas important for conformational flexibility, such as loops or ligand-binding domains (Supplementary Table 1).

The evolutionary conservation analysis supports the hypothesis that specific SPHK1 mutations may have functional consequences. Residues A186 and G369 lie within conserved domains across species, as shown by the multiple sequence alignment, highlighting their evolutionary importance. This conservation implies that substitutions at these positions could disrupt protein stability or function. In silico predictions using the Variant Effect Predictor (VEP), particularly SIFT and PolyPhen scores, reinforce these findings. Both A186T and G369S are classified as “damaging” with high confidence, suggesting potential deleterious effects (Table 2). The integration of conservation data and predictive tools provides robust support for the functional relevance of these variants, particularly in the context of cancer-associated alterations. This is consistent with prior studies highlighting the utility of conservation and in silico scoring in identifying biologically meaningful mutations (Ng & Henikoff, 2003; Adzhubei et al., 2010). The convergence of computational predictions and observed structural perturbations in the SPHK1 model underscores the potential impact of these mutations on enzymatic behavior and cancer progression [4-6].

The structural modeling conducted with AlphaFold and visualized in PyMOL (Supplementary Figure 2) provides complementary evidence. Regions harboring A186T and G369S mutations are located within highly confident pLDDT segments, suggesting these areas are well-defined structurally. The substitution of alanine and glycine with polar serine or threonine residues introduces steric and electrostatic shifts, particularly in compact or functionally relevant areas of the protein (Figure 2).

Given SPHK1’s role in converting sphingosine to sphingosine-1-phosphate (S1P), a lipid that promotes cancer cell proliferation and migration, subtle alterations in structure can have meaningful downstream effects [7]. Prior studies have shown that overexpression or hyperactivation of SPHK1 contributes to prostate cancer progression and may influence resistance to therapy [8-10]. Our findings suggest that mutations like A186T or G369S could represent regulatory adaptations, fine-tuning the enzyme’s function to meet altered metabolic needs in tumor environments.

Furthermore, the structural models generated (Figure 3) offer a reliable framework for hypothesizing the mechanistic consequences of each variant. While experimental validation is necessary, our data provide a foundation for designing site-directed mutagenesis or enzymatic activity assays in future work. These structural changes could affect substrate accessibility, catalytic efficiency, or interactions with regulatory lipids and proteins.