Fig. 3: Octameric assembly and molecular tunnels connecting the two active sites in hsNadE.

a The biological unit of hsNadE is generated by crystal symmetry as an octamer. The glutamine and ammonia tunnels in subunits A and B are in gray and golden, respectively. The red arrow indicates the direction of the substrate/product flows in/out the molecular tunnel. b, c Comparison of the constrictions located in the molecular tunnels found in hsNadE (b) and tbNadE-SFI complexes (c) within the single subunit A and two subunits D^glu–B^syn, respectively. Key residues (glutamine-binding residue, catalytic cysteine, and last position of the ammonia transport) are shown as spheres. d Polar, nonpolar, and charged residues forming the ammonia tunnel and constrictions in the hsNadE complex. The residues lining the ammonia tunnel belong to the glutaminase domain (green), the YRE loop (orange), and the synthetase domain (cyan). A water molecule coordinated with the residues forming constriction 1-1* and the YRE loop residues is shown in gray sphere with a light blue contour and its position indicates the start of the ammonia transport. e–g Ammonia tunnel radii calculated in subunit A of hsNadE complex (e), in subunits D^glu–B^syn (f), and in subunits C^glu-^symC^syn (g) of the tbNadE–SFI complex. The dashed line at constriction 1-1* shows the boundary of the glutamine and ammonia tunnels. 2.1 and 1.6 Å cut-off radii are used for the glutamine and ammonia tunnels, respectively. Location of the glutamine-binding site is defined by Tyr123^HS/Phe130^TB and Trp179^HS/Phe180^TB. The last position of ammonia in the ammonia tunnel is marked by Cys531^HS/Asp497^TB.