CryoEM analysis of human ACE dimer. (A) Diagram for the key features of domain on primary sequence of human ACE. To avoid confusion existing in the numbering of ACE structures, we number ACE based on Uniprot P12821-1. D1a and D3a domains, also called "lid" encompasses residues 31-127 and aa 645-725, respectively. D1b and D3b domains each have two discrete segments; residues 292-465 and 525-603 for D1b and residues 897-1060 and 1120-1200 for D3b. D2 encompasses three discrete segments, residues 128-291, 466-524, and 604-644 while D4 domain encompasses residues 726-896, 1061-1119, and 1201-1231. sp = signal peptide. asterisk = zinc binding motif. The construct used in this study comprises residues 1-1231, which we refer to as the soluble region of ACE (sACE). Colored by sub-domain: D1a cyan, D1b aqua, D2 pink, D3a light blue, D3b green, D4 magenta. (B) 2D classification of human sACE particles from grids made by vitrobot and Chameleon. Clear four domain classes visible in the Chameleon-derived classification are boxed in red, similar views are lacking in the vitrobot dataset. (C) Full-length sACE 3D volumes, colored by sub-domain as in (A). Glycan density is shown in gray. See Figure S2 for vitrobot-prepared data processing details, Figure S3 for Chameleon-prepared data processing details and Table S1 for data refinement statistics.

Overall structure of human ACE. (A) Overlay comparing sACE-N states highlighting the structure differences between the closed (C), intermediate (I), and open (O) states. (B) Overlay comparing sACE-C states highlighting the structure differences between the closed (C), and intermediate (I) states. We define the state based on the distance between the edge of the D2/4 domain bordering the catalytic cleft (residues 150-155 or 750-755) and the tip of the D1/3a region (residues 70-80 or 676-686): closed <15 Å, intermediate >15 Å and <19 Å, open >19 Å. (C) Overall dimer comparisons. (D) Table of openness measurements for each domain per structure.

sACE dimerization interfaces. (A) Overlay comparing sACE-N/N interface (blue) and sACE-C/C (yellow) interfaces. Interfaces adopt the same secondary structure but interacting residues vary between them. (B) Residue-specific interactions at the sACE-N/N interface, see text for details. (C) Unsharpened Coulomb potential density map (cyan) showing density corresponding to glycan-glycan interaction from N111 as part of sACE-N/N interface. Sharpened map is shown in magenta for reference. (D) Residue-specific interactions in the sACE-C/C interface, see text for details.

Structural mechanism of the sACE open/close transition. (A) sACE-N overlay comparing the open (colored in left panel, transparent gray in right panel) and closed (transparent gray in left panel, colored in right panel) states in detail. The open state is stabilized by interaction between residues in the D1a (cyan) and D2 (pink) regions that, notably K102-D218. In the closed state, the K102-D218 interaction is broken. (B) Overlay of the sACE-N open (cyan) and closed (pink) states showing the range of motion. The D1a region rotates about a fulcrum region described in (A), while the D1b region moves as a rigid body. (C) Overlay of sACE-C closed (light blue) and intermediate (yellow) states. Unlike sACE-N, the “top” of the D3a region is constrained by its connection to sACE-N and largely immobile. The primary source of opening is only the motion of the D3a tip. We did not observe any open state structures of sACE-C, suggesting a smaller range of motion relative to sACE-N. (D) Comparison of the hydrophobic “latch” region formed in the closed state between residues of the D1/3a, D1/3b, and D2/D4 domains. V753 in sACE-C has been replaced by T153 in sACE-N, suggesting that the closed state in sACE-N may be less stabilized than the sACE-C closed state. (E) Example all-atom MD simulation tracking the openness of one sACE-N region (black line) and this distance between K102 and D218 (red line). These residues form a salt bridge early in the simulation when sACE-N is open (left inset) but the interaction breaks as sACE-N transitions to the closed state (right inset). Distance measurements for MD simulations were consistently greater than distance values in our static structures and cannot be directly compared to Figure 2.

CryoEM heterogeneity analysis. (A) Visualization of the structural changes revealed by cryoSPARC 3DVA trajectories calculated along two principal components (PCs) of structural variance. Starting states are showing in cyan, ending states in gray. PC 0 reveals a large, inter-domain bending motion accompanied by the open/close transition in sACE-C. PC 1 and the remaining PCs are dominated by the open/close transition of individual regions. See Table S3, and Movie S7 for additional details. (B) Visualization of the structural changes revealed by the cryoDRGN trajectories calculated along two PCs of structural variance. Starting states are shown in cyan, ending states in gray. PCs are dominated by the open/close transition of individual regions. See Table S3, and Movie S8 for additional details. (C) Analysis in RECOVAR with a focus mask on sACE-N/N reveals that particles adopt roughly four clusters within the latent space (heat map of particle density) corresponding to the open-open (OO, white square), open-closed (OC, white dot), closed-open (CO, white dot) and closed-closed (CC, white star) states of sACE-N/N. A trajectory estimating the path through latent space corresponding to the structural transition from sACE-N/N CC state to the OO state (blue points) suggests that individual sACE regions transition at different rates. See Movie S9 for trajectory. (D) Focused 3D classification was performed in cryoSPARC to explore evidence of coordinated motion between sACE-N regions. 3D classification focusing on one sACE-N region revealed 2 roughly equal classes of particles: open and closed. Subsequent 3D classification focused on the other sACE-N region again revealed 2 roughly equal classes, suggesting the lack of coordinated motion between sACE-N regions in the absence of substrate.