Fig. 3

Molecular modeling of SRPK3 and subcellular localization of SRPK3 variants in HeLa cells. (A) Side chains of 3 amino acids involved in X-linked intellectual disability (XLID) are indicated as spheres on the SRPK3 structure modeled using AlphaFold. Unstructured loop regions of SRKP3 are omitted for clarity, including residues 1–46 and 310–385. (B) Interior misfolding due to H159D and T458N variations. SRPK3 wild-type (WT) left and the H159 and T458N variant form modeled based the AlphaFold structure prediction are shown together. (C) Structural superposition of the AlphaFold-modeled SRPK3 (green) onto the crystal structure of ICP27 (red)-bound SRPK1 (Navy; PDB code 6FAD). Side chain atoms of SRPK3 Glu529 are presented as spheres. (D) Alteration of protein shape and surface charge due to E529K variation. SRPK3 WT (left) and the E529K variant form (right) are shown as sticks and loops (top) or electrostatic surface representation (bottom). (E) Fluorescence cytochemistry images were obtained to compare the localization of SRPK3 WT and variants. Cell nuclei were stained with DAPI, and localization of GFP-SRPK3 was analyzed by observing GFP fluorescence using confocal microscopy. Scale bars; 20 μm. (F) Western blot analysis of GFP-SRPK3 was conducted to confirm the transfection efficiency of WT and variant plasmids. GAPDH served as a loading control.