Fig. 1

a Domain organization of ARF3 excluding the unstructured C-terminal tail. Switch 1, switch 2 and the NKXD fingerpoint motif are highlighted in pink, green and yellow, respectively. The variants identified in affected subjects are also reported. b 3D structure in two different orientations of GTP-bound ARF3 interacting with the MARTX toxin (PDB 6ii6). Side chains of the ARF3 residues mutated in the affected subjects and GTP are in cyan and red, respectively. Main chain of residues belonging to switch 1, switch 2 and NKXD fingerpoint motif are colored as above. c Enlargement of the ARF3 GTP binding pocket with the five mutated residues. The direct hydrogen bond between the N atom in the Lys127 lateral chain and the oxygen atom of the GTP ribose ring is highlighted in dashed line. The Mg²⁺ ion is colored in magenta, while the oxygen atom of the water molecule, mediating the interaction between Asp⁶⁷ and the manganese ion, is shown in light blue color. The two hydrogen bonds between Asp⁶⁷ and the water molecule are highlighted with dotted lines. d Zoom showing the structural organization around residue 93. Left: view of the WT Asp⁹³ forming a hydrogen bond with Lys¹²⁷. Right: structure with the p.Asp93Asn mutation and hydrogen bond breaking. The Mg²⁺ ion is colored in magenta. e Homology model of GTP-bound ARF3 interacting with the cytosolic coat protein complex COPG1-COPZ1 (PDB: 3TJZ) validated by a 500-ns molecular dynamics (MD) simulation. The region of contact between ARF3 and COPG1 (orange color) is shown in (e). f?h MD simulations of wild-type (f), p.Asp67Val (g), and p.Pro47Ser (h) ARF3 complexed with COPG1-COPZ1. Residues involved in the contact are shown with their side chain and colored as the respective protein/region. ARF3 backbone is represented with a diameter proportional to its per-residue fluctuations (RMSF).