Fig. 3: Stabilization and inactivation of the enzymatic activity of T3A.

a Melting temperatures measured via DSF for wildtype monomeric ACE2 (wt, black line) and monomeric ACE2 with N51C–V343C or G339C–T517C (magenta and teal, respectively), n = 2. b A representative cartoon model of monomeric ACE2 (PDB ID: 6M0J) with introduced N51C–V343C disulfide bond highlighted (red sticks); model is colored pink with terminals labeled blue (N-) and red (C-). c, d Ribbon diagram and X-ray diffraction 2Fo-Fc electron density maps at 1.0 s highlighting the introduced disulfide bond: c E31C–V'37C or d G(-1)C–L'5C; carbons are colored from N- (blue) to C-terminus (red) for a single chain, and gray for the other two chains. e, f Melting temperatures measured via DSF of e wt collagen XVIII trimerization domain (black line) and disulfide mutants: G22C–F'26C, E31C–V'37C, G(-1)C–L'5C (magenta, teal, purple, respectively), n = 2, and f unmutated T3A and disulfide mutants: T3A^D (N51C–V343C in ACE2), T^D3A (E31C–V'37C in trimerization domain), T^D3A^D (E31C–V'37C in trimerization domain and N51C–V343C in ACE2), n = 2. g Enzymatic activity measured via cleavage of fluorescence peptide substrate analog with trendline depicting mean RFU, n = 2, and h melting temperature measured via DSF for T3A, T^D3A^D, T^D3A^D_R273Q, and T^D3A^D_H345F, n = 3. i T3A constructs incubated in rat serum at 37 °C were sampled over 216 h via ELISA using a SARS-CoV-2 spike HexaPro protein-coated plate. An HRP-conjugated anti-Myc tag was used to determine binding to spike protein with trendline fit to a single phase decay, n = 4. j MD simulations depicting RMSD (in Å) between 150–400 ns for wt ACE2 (black) and ACE2 with mutations: N51C–V343C (magenta); N51C–V343C with R273Q (purple); and N51C–V343C with H345F (teal), n = 1. Errors described by standard deviation from n biological replicates. All melting, activity, and stability curves generated from a single, representative experiment for clarity.