Engineering human ACE2 to optimize binding to the spike protein of SARS coronavirus 2

Abstract

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds angiotensin-converting enzyme 2 (ACE2) on host cells to initiate entry, and soluble ACE2 is a therapeutic candidate that neutralizes infection by acting as a decoy. By using deep mutagenesis, mutations in ACE2 that increase S binding are found across the interaction surface, in the asparagine 90-glycosylation motif and at buried sites. The mutational landscape provides a blueprint for understanding the specificity of the interaction between ACE2 and S and for engineering high-affinity decoy receptors. Combining mutations gives ACE2 variants with affinities that rival those of monoclonal antibodies. A stable dimeric variant shows potent SARS-CoV-2 and -1 neutralization in vitro. The engineered receptor is catalytically active, and its close similarity with the native receptor may limit the potential for viral escape.

Fig. 1. Sequence preferences of ACE2 residues for high binding to the RBD of SARS-CoV-2 S.

(A) Log₂ enrichment ratios from the nCoV-S-High sorts are plotted from depleted or deleterious (orange) to enriched (dark blue). ACE2 primary structure is shown on the vertical axis, amino acid substitutions are indicated on the horizontal axis. Wild-type amino acids are in black. Asterisk (*) denotes stop codon. (B) Conservation scores are mapped to the structure (Protein Data Bank 6M17) of RBD (green ribbon)–bound protease domain (surface), oriented with the substrate-binding cavity facing the reader. Residues conserved for RBD binding are shown in orange; mutationally tolerant residues are in pale colors; residues that are hot spots for enriched mutations are in blue; and residues maintained as wild type in the ACE2 library are in gray. Glycans are depicted as dark red sticks. (C) Viewed looking down on to the RBD interaction surface. (D) Average hydrophobicity-weighted enrichment ratios are mapped to the structure, with residues tolerant of polar substitutions in blue and residues that prefer hydrophobics in yellow. (E) A magnified view of the ACE2–RBD interface [colored as in (B) and (C)]. Heat-map plots log₂ enrichment ratios from the nCoV-S-High sort. Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

Fig. 2. A variant of sACE2 with high affinity for S.

(A) Expi293F cells expressing S were incubated with purified wild-type sACE2 (gray) or sACE2.v2 (blue) fused to 8His (solid lines) or IgG1-Fc (broken lines). Bound protein was detected by flow cytometry. Data are mean fluorescence units (MFU) of the total cell population after subtraction of background autofluorescence. n = 2 replicates, error bars represent range. (B) Binding of 100 nM wild-type sACE2-IgG1 (broken lines) was competed with wild type sACE2-8h (solid gray line) or sACE2.v2-8h (solid blue line). The competing proteins were added simultaneously to cells expressing S, and relative bound protein was detected by flow cytometry. n = 2 replicates, error bars represent range. (C) Competition for binding to immobilized RBD in an ELISA between serum IgG from COVID-19 patients versus wild-type sACE2-8h (gray) or sACE2.v2-8h (blue). Three different patient sera were tested (P1 to P3 in light to dark shades). Data are mean ± SEM, n = 2 replicates.

Fig. 3. A dimeric sACE2 variant with improved properties for binding viral spike.

(A) Analytical SEC of wild-type sACE2₂-8h (gray) and sACE2₂.v2.4-8h (purple) after incubation at 37°C for 62 hours. (B) ELISA analysis of serum IgG from COVID-19 patients (P1 to P3 in light to dark shades) binding to RBD. Dimeric sACE2₂(WT)-8h (gray) or sACE2₂.v2.4-8h (purple) are added to compete with antibodies recognizing the receptor-binding site. Concentrations are based on monomeric subunits. Data are mean ± SEM, n = 2 replicates. (C) RBD-8h association (t = 0 to 120 s) and dissociation (t > 120 s) with immobilized sACE2₂(WT)-IgG1 measured by BLI. (D) BLI kinetics of RBD-8h binding to immobilized sACE2₂.v2.4-IgG1.

Fig. 4. Enhanced neutralization of SARS-CoV-1 and -2 by engineered receptors.

In a microneutralization assay, monomeric (solid lines) or dimeric (broken lines) sACE2(WT)-8h (gray) or sACE2.v2.4-8h (purple) were preincubated with virus before adding to VeroE6 cells. Concentrations are based on monomeric subunits. Data are mean ± SEM of n = 4 replicates.