Fig. 3

Protein alignment with location of RAX2 variants and RAX2 structural analysis. a Amino acid alignment of RAX2 orthologues. Amino acid sequences deduced from the nucleotide sequences were downloaded from the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/). The accession numbers of the sequences used in this alignment study are as follows. Homo sapiens: hs_rax2, NM_032753; Pan troglodytes: pt_rax2, NM_001081487; Macaca mulatta: mm_rax2, XM_001100945; Bos taurus: bt_rax2, NM_182653; Gallus gallus: gg_rax2, AF420601; Xenopus laevis: xl_rxL, DQ360108; Danio rerio: dr_rx1, AF001907; Danio rerio: dr_rx2, AF001908. The alignment was performed using the MUSCLE algorithm with default parameters in the Unipro UGENE v1.29.0 software. Gaps required for optimal alignment are indicated by dashes. The color intensity of the amino acids is according to percentage identity. The highly conserved RAX2 homeodomain is indicated by a black bar. The three RAX2 pathogenic sequence variants identified in this study are indicated at the amino acid level in black. The four previously reported RAX2 pathogenic variants involved in autosomal dominant cone-dominated retinal disease are indicated at the amino acid level in gray. b Homology modeling of the RAX2 homeodomain. Structure superposition of a homology model of the RAX2 homeodomain with six homeodomain crystal structures. The loop between helix 1 and 2, containing Ser49 and Pro52, is structurally conserved. Protein Data Bank (PDB) codes of superposed structures are 3A01_A, 3A01_B, 1FLJ_A, 2H1K_A, 1IG7_A, 5Z2T_C. c Model for RAX2 and CRX bound to a Ret-1 fragment. In the model, Arg87 of RAX2 interacts with Gln70 of CRX. PDB codes of structure used as template: 3A01_A. d Model with the N-terminal extension of RAX2. In this model, a positively charged region inserts in the minor groove. The RAX2 homeodomain and its N-terminal domain completely encircle the DNA fragment, and the ring is closed by a Glu12-Arg87 salt bridge. The sequence of the positively charged region in RAX2 and its counterpart in the clawless template are shown. RAX2 residue Glu12 and clawless site 1 residue (Arg171) are underlined. Green: negatively charged amino acids. Blue: positively charged amino acids.