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Fig. 1 Molecular characterization of Xenotoca eiseni voltage-sensing phosphatase (Xe-VSP). A: schemes of the domain structures of X. eiseni VSP (Xe-VSP), zebrafish VSP (Dr-VSP), and human VSP1 (Hs-VSP1), demonstrating the conservation of functional domains across different species. Multiple amino acid alignment of full-length VSP orthologs is provided in Supplemental Fig. S1. B: predicted model of Xe-VSP, highlighting its key functional domains: transmembrane voltage-sensing domain (VSD) (gray), VSD-phosphatase domain (PD) linker (green), phosphatase domain (blue), and C2 domain (magenta). The average predicted local-distance difference tests (pLDDT) score = 87.2. C: amino acid sequence comparison of transmembrane VSDs, VSD-PD linkers, and Cx5R(T/S) motifs in the PDs of Xe-VSP and Dr-VSP. Symbolic conservation scores are indicated below the aligned sequences. Note that the S4 segments of both VSPs contain multiple positively charged arginine (R, highlighted in blue) intervened by hydrophobic residues. The Cx5R(T/S) motif, crucial for phosphatase enzyme activity, is highly conserved among VSPs. Red highlights a cysteine residue (C) within the active center of the PD that is required to form a cysteinyl-phosphate intermediate, a critical step in substrate dephosphorylation. D: phylogenetic tree showing the evolutionary relationships among VSP orthologs. Data were retrieved from NCBI public database and analyzed using the neighbor-joining method in MEGAX software. Ci, Ciona intestinalis Type A; Cp, Cynops pyrrhogaster; Dr, Danio rerio; Gg, Gallus gallus; Hn, Hynobius nebulosus; Hs, Homo sapiens, Mm, Mus musculus; Pr, Poecilia reticulata; Rn, Rattus norvegicus; Xe, Xenotoca eiseni; Xl, Xenopus laevis