Angiotensin‐converting enzyme open for business: structural insights into the subdomain dynamics

Figures

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  Fig. 1. Schematic structure representations. Crystal structures of (A) typical closed nACE (PDB code‐6F9V [32]). (B) open nACE. (C) Open S2_S′‐nACE and (D) native cACE. The active‐site zinc ion is depicted as a lilac sphere, zinc‐binding residues (His‐, His‐ and Glu‐) as sticks, with α‐helices and β‐strands (in panels B–D) coloured rose and dark cyan, respectively. Panel A shows subdomains 1 and 2 in orange (the lid‐like region in dark orange) and green colour, respectively. Loop regions have been smoothed for clarity. Figure was generated using CCP4mg [47].

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  Fig. 2. Surface representation of the open nACE structure. Two views are shown rotated 90° about the x‐axis, with the peptide chain coloured from blue (N terminus) to red (C terminus) as indicated by the cylinder. This shows that the two subdomains have opened to allow access to the binding site cavity. Figure was generated using CCP4mg [47].

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  Fig. 3. Variation in opening of molecules on nACE in the asymmetric unit. The four molecules in the open nACE structure asymmetric unit are overlaid using the subdomain 2 residues. This highlights differences in the subdomain 1 overlay indicating a small variation in degree of opening. Subdomains 1 and 2 are shown in lighter and darker colours, respectively. Figure was generated using CCP4mg [47].

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  Fig. 4. Open nACE structure active site. Close‐up view of the zinc and substrate C terminus binding sites of the nACE open structure. The zinc ion interacts with the conserved two histidine and one glutamate residues, with the coordination sphere completed by a water molecule that also binds to the catalytic Glu‐362. The substrate C terminus binding site is occupied by a calcium ion coordinated by two bicine molecules. α‐helices and β‐strands coloured rose and dark cyan, respectively. Figure was generated using CCP4mg [47].

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  Fig. 5. Novel cACE structure with C terminus insertion. A native G13‐cACE crystal structure shows the insertion of a symmetry‐related molecule's C terminus into the nonprime lobe and interaction with the active‐site zinc ion. Zinc ions are shown as lilac spheres, with α‐helices and β‐strands shown in rose and dark cyan, respectively. (A) Close‐up view of the C terminus insertion overlaid with the final 2mFo –DFc (blue, contoured at 1σ level) electron density map. (B) Arrangement of G13‐cACE crystal packing showing molecule A (green worm) with its C terminus orientated to insert into the nonprime lobe of symmetry‐related molecule B. (C) Close‐up view of C terminus insertion showing interacting residues of molecule A (green worm—residues 619–633 were used to generate figure, but not all residues are visible within the cavity) at the surface of symmetry‐related molecule B. (D) Native G13‐cACE LigPlot representation showing the binding of the C terminus of molecule A (dark green) within the nonprime binding lobe of symmetry‐related molecule B (light green). H‐bond/electrostatic interactions are shown in green, hydrophobic interactions in red and water molecules as red spheres. Residues…

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  Fig. 6. Schematic representation showing structural overlays. Structural overlays of (A) closed nACE (blue) and open (green) nACE, (B) closed nACE (blue), closed cACE (orange) and closed ACE2 (grey), (C) open nACE (green) and open ACE2 (magenta) and (D) open nACE (green), open cACE (dark red) and open ACE2 (magenta). Zinc atoms are shown as spheres and coloured lighter than their corresponding protein. α‐helices and β‐strands are depicted as cylinders and arrows, respectively, with loop regions shortened for clarity. PDB codes for closed nACE (sampatrilat complex), closed cACE (omapatrilat complex), closed ACE2 (MLN‐4760 complex), open cACE (BPPb complex) and open ACE2 (native) are 6F9V [32], 6H5W [37], 1R4L [26], 4APJ [34] and 1R42 [26], respectively. Figure was generated using CCP4mg [47].

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  Fig. 7. Surface representation comparison of ACE/ACE2 opening. Surface representations of (A) closed nACE, (B) BPPb‐cACE, (C) open nACE and (D) ACE2 structures. The peptide chains are coloured from blue (N terminus) to red (C terminus) as indicated by the cylinder, and the active‐site zinc (if visible) is a lilac sphere. PDB codes for closed nACE (sampatrilat complex), open cACE (BPPb complex) and open ACE2 (native) are 6F9V [32], 4APJ [34] and 1R42 [26], respectively. Figure was generated using CCP4mg [47].

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  Fig. 8. N389‐nACE sequence.Sequence and secondary structure (generated using STRIDE [50] from sampatrilat complex PDB 6F9V [32]) of nACE. Helices (H) are numbered sequentially, β‐strands and turns are indicated in yellow and green, respectively, and hinges are shown in blue boxes.

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  Fig. 9. Mechanism of nACE opening. Overlay of the open and closed nACE structures highlighting the regions that move during the domain opening. Each moving region is also shown in isolation for clarity with the largest movement point marked. Subdomain 2 is shown in dark green/light green, with the moving regions of subdomain 1 coloured dark red/coral (residues 11–97), orange/yellow (residues 272–378), magenta/light pink (residues 384–417), dark blue/light blue (residues 507–547) and dark grey/light grey (residues 552–566). Zinc ion is shown as violet/lilac sphere. Darker colours are the closed structure. Figure was generated using CCP4mg [47].

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  Fig. 10. Comparison of the subdomain interface region. Close‐up view of the subdomain interface region of (A) typical closed nACE structure (PDB code‐6F9V [32]), (B) overlay of the four molecules in the open nACE crystal structure asymmetric unit (zinc ion and waters of only one molecule shown for clarity) and (C) overlay of the four molecules in the open S2_S′‐nACE crystal structure asymmetric unit. S2′ and distal S′‐residues are coloured green and magenta, respectively. Figure was generated using CCP4mg [47].

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  Fig. 11. ACE structural adaptations for substrate promiscuity. (A) Flexibility of lid‐like region adjacent to C terminus insertion. Two views of the overlay of samASP‐nACE and C terminus insertion cACE structures showing how the flexibility observed in the lid‐like region has an effect on hole at the bottom of the nonprime lobe and could interact with peptides passing through this hole. The samASP‐nACE alternate lid‐like conformations are shown in light and dark blue, with cACE in orange. The C terminus of symmetry‐related cACE molecule is shown as green worm. (B and C) Accommodation of variation in peptide substrate size in ACE. Schematic representation of an overlay of nACE open structure with the BPPb peptide (yellow sticks) from an nACE complex structure (PDB 6QS1 [35]) showing (B) the hole at the base of the nonprime lobe, and (C) highlighting the possible routes for extension of the prime binding lobe. Binding groove/cavity is shown in grey with the active‐site zinc as magenta sphere. Panel A was generated using CCP4mg [47], with B and C using pymol [48].