Fig. 3

Structural modeling and in-silico analysis of the effect of missense variants identified in NOS3 and GUCY1A3. A and F: Multiple Sequence Alignments (MSA) showing that the residues C648 of eNOS and R593 of alpha sCG are evolutionary conserved residues. B and C: 3D-structures of the wild type eNOS protein (experimental and 3D models). The C648 residue is located in the reductase domain of the protein. D and E: A zoom in the region of the C648 residue is presented in D (wild-type protein) and E (mutated p.C648R protein). The substitution p.C648R is predicted to destabilize the 3D-structure of the domain (DUET prediction ΔΔG = − 1.0 kcal/mol). There is no space to accommodate the larger and potentially positively charged arginine side chain in the region of the protein (steric clashes are represented by red and green cylinders).G and H: 3D-structures of the wild type sCG protein in an inactive (G) and NO-activated (H) states. sCG is a heterodimer composed of an α-subunit and a β-subunit. The R593 residue mutated in M038 is carried by the α-subunit and is located in the catalytic domain of sCG protein. I and J: A zoom in the region of the R593 residue is presented in I (wild-type inactive state) and J (wild-type NO-activated state). R593 takes part in a salt-bridge network that involves the evolutionary conserved residues E526 (α -subunit), R539 and E473 (β-subunit). E473 interacts with the GTP substrate when the sCG is activated (GMPCPP: GTP binding pocket). The substitution p.R593H is predicted to destabilize the 3D-structure of the inactive (ΔΔ G = -1.5 kcal/mol) and active forms (ΔΔG = − 1.60 kcal/mol) of sGC, through perturbation of non-covalent interactions in the catalytic domain, and negatively impact the formation of the GTP binding pocket