key: cord-0933729-6h6dmbl1 authors: Guo, Liang; Bi, Wenwen; Wang, Xinling; Xu, Wei; Yan, Renhong; Zhang, Yuanyuan; Zhao, Kai; Li, Yaning; Zhang, Mingfeng; Bao, Xingyue; Cai, Xia; Li, Yutang; Qu, Di; Jiang, Shibo; Xie, Youhua; Zhou, Qiang; Lu, Lu; Dang, Bobo title: Engineered Trimeric ACE2 Binds and Locks “Three-up” Spike Protein to Potently Inhibit SARS-CoVs and Mutants date: 2020-09-01 journal: bioRxiv DOI: 10.1101/2020.08.31.274704 sha: c6603118730f5f22fc2267352883becc075e75e1 doc_id: 933729 cord_uid: 6h6dmbl1 SARS-CoV-2 enters cells via ACE-2, which binds the spike protein with moderate affinity. Despite a constant background mutational rate, the virus must retain binding with ACE2 for infectivity, providing a conserved constraint for SARS-CoV-2 inhibitors. To prevent mutational escape of SARS-CoV-2 and to prepare for future related coronavirus outbreaks, we engineered a de novo trimeric ACE2 (T-ACE2) protein scaffold that binds the trimeric spike protein with extremely high affinity (KD < 1 pM), while retaining ACE2 native sequence. T-ACE2 potently inhibits all tested pseudotyped viruses including SARS-CoV-2, SARS-CoV, eight naturally occurring SARS-CoV-2 mutants, two SARSr-CoVs as well as authentic SARS-CoV-2. The cryo-EM structure reveals that T-ACE2 can induce the transit of spike protein to “three-up” RBD conformation upon binding. T-ACE2 thus represents a promising class of broadly neutralizing proteins against SARS-CoVs and mutants. motif and ACE2 would be around 60 Å, which corresponds to the length of the (GGGGS)3 154 linker. Thus, the (GGGGS)5 flexible linker in some of our designed proteins is long enough for 155 three ACE2s to bind, but is not optimal. The more rigid (EAAAK)5 linker is shorter than 156 (GGGGS)5 and can effectively separate different functional domains of fusion proteins 46 . We 157 think the effective length of the (EAAAK)5 linker is probably around 60 Å, making it an optimal 158 linker for T-ACE2. The rigid nature of this (EAAAK)5 linker probably helps to orient ACE2 159 right around RBD for immediate rebinding even if one ACE2 monomer from T-ACE2 160 dissociates from the spike protein. we engineered trimeric ACE2 proteins based on wild-type ACE2 and showed that T-ACE2 could 176 bind spike protein with extremely high affinity to potently inhibit all tested pseudotyped viruses 177 including SARS-CoV-2, SARS-CoV, eight naturally occurring SARS-CoV-2 mutants, two 178 SARSr-CoVs as well as authentic SARS-CoV-2. The rigid linker employed in T-ACE2 was 179 previously injected into mice and didn't seem to show strong immunogenicity 50 . The 3HB and 180 foldon trimerization motifs have been observed to cause immunogenicity, but the introduction of 181 glycans could silence the immunogenicity without disrupting the trimer formation 51 . Carrying 182 these advancements a few steps beyond, the modular design of T-ACE2 demonstrates that other 183 oligomerization motifs and linkers could be further explored to improve properties of T-ACE2 or 184 higher oligomeric ACE2s. 185 We demonstrated that T-ACE2 could induce the transit of spike protein to a unique "three-186 up" RBD conformation and bind all three RBDs simultaneously. Whether this T-ACE2-induced 187 spike protein conformation change represents a transition state during virus infection cannot be 188 definitively answered here. Full-length ACE2 protein functions as a dimer 42 , the two monomers 189 from this ACE2 dimer are related by two-fold symmetry. They are also situated close in space, 190 with the distance between D615 being about 53 Å. Thus, the native dimeric ACE2 is unlikely to 191 engage more than one RBD from the same spike protein without substantial conformational 192 changes. It is however possible that ACE2 dimers on the cell surface might further cluster to 193 induce more RBDs to adopt up conformation and help virus to transit from the prefusion state to though further studies are still needed to confer this advantage. Thus, proteins engineered based 202 on wild-type ACE2, such as T-ACE2, can potently and broadly inhibit virus infections, they also 203 have the added benefits of regulating RAS and alleviating ARDS. These potential beneficial 204 effects distinguish proteins like T-ACE2 from neutralizing antibodies. We believe T-ACE2 205 represents a promising class of proteins to broadly inhibit SARS-CoVs and to treat viruses 206 infected patients. Finally, the extremely high binding affinity between T-ACE2 and spike protein 207 (KD < 1pM) suggests that T-ACE2 could also be useful for virus detection. The fact that T-ACE2 208 was engineered based on native ACE2 sequence also makes such detection methods widely 209 useful for all SARS-CoVs and related viruses. To construct trimeric ACE2s, we inserted the linker sequences (GGGGS)5, (EAAAK)5 or 290 GGGS after ACE2(1-615), followed by trimerization motifs, an HRV3C cleavage sequence, an 291 eGFP tag and a His8 tag. Monomeric ACE2 was constructed as ACE2(1-615)-(GGGGS)5-292 HRV3C-eGFP-His8 for direct comparison. For purification of ACE2 proteins, the cell supernatants were harvested by centrifugation at 306 1000×g for 5 minutes. Then the supernatants were loaded on Ni-NTA beads (Smart-Lifesciences, 307 Cat. SA004100) and washed with washing buffer (5 mM imidazole, 1 × PBS). Proteins were 308 then eluted with elution buffer (50 mM imidazole, 1 × PBS). 309 The eluted proteins were concentrated and subjected to size-exclusion chromatography 310 (Superose 6 Increase 10/300 GL, GE Healthcare) in the PBS buffer. The peak fractions were 311 collected and concentrated. The proteins were then analyzed by size exclusion chromatography 312 (AdvanceBio SEC 300Å) in PBS buffer pH 7.4. The standard proteins were purchased from GE 313 S2) . 314 To remove C-terminal tags of ACE2 proteins, 16 µg HRV3C protease (expressed and 315 purified in house) was add to 1mg ACE2 protein and incubated at 4 °C overnight, followed by For kinetics analyses, S-ECD was captured on streptavidin biosensors. Biotinylated S-ECD 361 was diluted to 20 µg/mL in dilution buffer (PBS with 0.02% Tween 20 and 0.1% BSA). Then 362 sensor baselines were equilibrated in the dilution buffer for 90 seconds. Next, the S-ECD was 363 loaded until the thickness signal was 0.6 nm or 0.3 nm (low loading). After loading, the sensors 364 were washed for 60 seconds in the dilution buffer. The sensors were then immersed into wells 365 containing ACE2 proteins for 100 seconds (association phase), followed by immersion in 366 dilution buffers for an additional 300 seconds (dissociation phase). The background signal was 367 measured using a reference sensor with S-ECD loading but no ACE2 protein binding and was 368 subtracted from the corresponding ACE2 binding sensor. Curve fitting was performed using a The live SARS-CoV-2 inhibition assay was performed as previously described 56 . Briefly, 412 Vero-E6 cells were seeded into the 96-well cell culture plate at 3×10 4 per well and cultured for 413 12 hours. Recombinant proteins were diluted with FBS-free DMEM, mixed with 100 TCID50 of 414 SARS-CoV-2, and incubated at 37 ℃ for 1 hour. Then, the protein-virus mixtures were added to 415 Vero-E6 cells and incubated at 37 ℃ for 1 hour. After removing the mixtures, cells were 416 cultured with fresh DMEM containing 2% FBS for another 48 hours. Then, the supernatants 417 were collected to detect viral RNA titer. The cells were fixed to perform immunofluorescence 418 analysis. After fixing with 4% paraformaldehyde, the cells were permeabilized by 0.2% Triton 419 X-100 and blocked with 3% BSA for 1 hour. Then the SARS-CoV-2 Nucleocapsid Antibody 420 (1:500) (Sino Biological) was added to cells and reacted at 4℃ overnight. Finally, the cells were Particles were automatically picked using Relion 3.0.6 62-65 from manually selected 462 micrographs. After 2D classification with Relion, good particles were selected and subjected to 463 two cycles of heterogeneous refinement without symmetry using cryoSPARC 66 .The good 464 particles were selected and subjected to Non-uniform Refinement (beta) with C1 symmetry, 465 resulting in the 3D reconstruction for the whole structures that were further subjected to 3D 466 classification, 3D auto-refinement and post-processing with Relion. For interface between RBD 467 and ACE2, the datasets were subjected to focused refinement with adapted mask on each RBD 468 and ACE2 subcomplex to improve the map quality. Then the dataset of three RBD and ACE2 469 sub-complexes were combined and subjected to focused refinement with Relion, resulting in the 470 3D reconstruction of better quality on the interface between S-ECD and ACE2. 471 The resolution was estimated with the gold-standard Fourier shell correlation 0.143 criterion 472 67 with high-resolution noise substitution 68 . Refer to (fig S6-8) and Supplemental Table S1 for 473 details of data collection and processing. geometry restraints to prevent overfitting. To monitor the potential overfitting, the model was 485 refined against one of the two independent half maps from the gold-standard 3D refinement 486 approach. Then, the refined model was tested against the other map. Statistics associated with 487 data collection, 3D reconstruction and model building were summarized in Table S1 . 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