Bioinformatic Analysis of WNT Family Proteins

Abstract

WNT proteins constitute a highly conserved family of signaling molecules that play an important role in regulating embryonic development and maintaining adult tissue homeostasis. Their diverse biological functions are mediated through multiple signaling pathways, including both canonical β-catenin-dependent and several non-canonical mechanisms. The regulatory activity of WNT proteins is closely linked to their unique structural organization, the presence of N-terminal signal peptides, and posttranslational modifications. In this study, in silico methods were used to analyze the structural features of WNT proteins. Specifically, the isoelectric points, GRAVY scores, aliphatic indices, and instability indices were determined, and correlation analysis was performed to examine relationships between the latter three parameters. In addition, the characteristics of N-terminal signal peptides in WNT family proteins were investigated, with a particular focus on the bioinformatic prediction of N-terminal peptide lengths in the WNT2B protein isoforms. Furthermore, in silico modeling and molecular dynamics simulations were employed to study the tertiary structure of WNT2B and to assess the significance of O-acylation at serine for the behavior of the mature protein in an aqueous environment. Thus, using computational approaches, new data were obtained on the structural features and dynamic properties of this group of regulatory proteins.

Keywords: WNT proteins; acylation; in silico analysis; molecular dynamics; posttranslational modifications; signal peptides.

Figure 1.

Distribution of aliphatic index, GRAVY score, and instability index in WNT-related proteins. (A) 3D scatterplot illustrating the relationship between the 3 indices; (B) scatterplot showing the correlation between GRAVY and the aliphatic index; (C) scatterplot showing the correlation between GRAVY and the instability index; (D) scatterplot showing the correlation between the instability index and the aliphatic index. The color gradient represents the length of amino acid sequences in WNT proteins.

Figure 2.

Prediction of the lengths of the N-terminal signal peptide sequences in Q93097-1 (A) and Q93097-2 (B) isoforms of WNT2B using SignalP 5.0 tool.

Figure 3.

WNT2B models generated by different tools: AlphaFold3 (A), RoseTTAFold (B), SWISS-MODEL (C) D-I-TASSER (D), DMPfold2 (E).

Figure 4.

Analysis of molecular dynamics trajectories of WNT2B. (A) root-mean-square deviation (RMSD), (B) root-mean-square fluctuation (RMSF), (C) radius of gyration (Rg), (D) solvent-accessible surface area (SASA). Graphs for non-acylated WNT2B are presented in blue, and for O-acylated WNT2B in green.

Figure 5.

Structural alignment of WNT2B models at 100 ns (A) and 200 ns (B). Non-acylated WNT2B is shown in blue, and O-acylated WNT2B in green.

Figure 6.

Principal component analysis (PCA) and free energy landscape (FEL) analysis for molecular dynamics of WNT2B. (A-C) PCA plots for 2 models of WNT2B simultaneously (A), non-acylated WNT2B (B), and O-acylated WNT2B (C). (D-E) FEL plots for non-acylated (D) and O-acylated (E) WNT2B. Dots corresponding to non-acylated WNT2B are shown in blue on PCA plots, and for O-acylated WNT2B in green.