key: cord-0786822-r9di6z2w
authors: Low-Gan, Jessie; Huang, Ruiqi; Warner, Gabrielle; Kelley, Abigail; McGregor, Duncan; Smider, Vaughn
title: Diversity of ACE2 and its interaction with SARS-CoV-2 receptor binding domain
date: 2020-11-04
journal: bioRxiv
DOI: 10.1101/2020.10.25.354548
sha: e5b9c92fe527275f661ef11be9af4013a051cabb
doc_id: 786822
cord_uid: r9di6z2w

COVID-19, the clinical syndrome caused by the SARS-CoV-2 virus, has rapidly spread globally causing tens of millions of infections and over a million deaths. The potential animal reservoirs for SARS-CoV-2 are currently unknown, however sequence analysis has provided plausible potential candidate species. SARS-CoV-2 binds to the angiotensin I converting enzyme 2 (ACE2) to enable its entry into host cells and establish infection. We analyzed the binding surface of ACE2 from several important animal species to begin to understand the parameters for the ACE2 recognition by the SARS-CoV-2 spike protein receptor binding domain (RBD). We employed Shannon entropy analysis to determine the variability of ACE2 across its sequence and particularly in its RBD interacting region, and assessed differences between various species’ ACE2 and human ACE2. As cattle are a known reservoir for coronaviruses with previous human zoonotic transfer, and has a relatively divergent ACE2 sequence, we compared the binding kinetics of bovine and human ACE2 to SARS-CoV-2 RBD. This revealed a nanomolar binding affinity for bovine ACE2 but an approximate ten-fold reduction of binding compared to human ACE2. Since cows have been experimentally infected by SARS-CoV-2, this lower affinity sets a threshold for sequences with lower homology to human ACE2 to be able to serve as a productive viral receptor for SARS-CoV-2.

SARS-CoV-2 utilizes its trimeric spike protein to bind to the angiotensin I converting enzyme 2 (ACE2) 65 on target pneumocytes or other host cells (22) (23) (24) (25) (26) (27) (28) . This interaction occurs with high affinity, and results 66 in viral membrane fusion to the host cell and initiates the infectious process. SARS-CoV-1 also utilizes 67 ACE2 as a receptor, however their spike proteins bind with lower affinity (31 nM KD) than SARS-CoV-2 68 (4.2 nM KD) (23) . Crystal structure and electron microscopy analysis of SARS-CoV-2 with ACE2 has 69 revealed the interacting amino acid residues of the spike receptor binding domain (RBD) and the 70 human ACE2 surface (23, 25, 29, 30) . With sequences available for many companion and agriculturally 71 important species, the ability to assess potential spike RBD binding is an important first step towards 72 prediction of infection of these alternative hosts. Here we analyze the conservation and diversity of the 73 ACE2 protein in multiple important animal species, with particular emphasis on the region that interacts 74 with SARS-CoV-2 spike RBD. We confirm that an ACE2 from Bos taurus, which is somewhat divergent 75 from human ACE2 in its binding region, interacts with SARS-CoV-2 spike RBD with high affinity, 76

suggesting that multiple mammalian species may be susceptible to infection with this coronavirus. 77

Results 79

In order to assess the potential of SARS-CoV-2 to interact with ACE2 of important companion and 80 agricultural species, we assembled the ACE2 sequences of several species (Table 1 and Supplemental  81   Table 1 ). The human ACE2/RBD cocrystal structure (PDB: 6M17)(27) was utilized to visualize 82 interacting residues between the SARS-CoV-2 spike RBD and human ACE2 (Figure 1 ). In ACE2, 8 83 residues on two helices make direct contact with spike RBD and an additional 17 residues are within 5 84 angstroms of the RBD. These 25 residues are color coded in Figure 1 as purple (contact) and cyan 85 (nearby), respectively. We focused our evaluation on these interacting residues in the following 86 analyses. 87

In order to determine the overall differences between ACE2 of different species, we employed protein 88 sequence alignment as well as variability analysis (Supplemental Figures 1 and 2 ). All vertebrate 89 ACE2 sequences showed significant homology to human ACE2, with cynomologous and rhesus 90 monkeys being 95% and 94% identical to human (Table 1) . Other mammals were between 80-87% 91 identical to human. Horseshoe bats, a potential reservoir for SARS-CoV-2 (28,31) was only 81% and 92 76% identical through the entire sequence and interacting residues, respectively. Surprisingly, the 93 percent identity across the entire ACE2 sequence did not fully correlate with percent identity of the 94 interacting residues. For example, cows show lower homology throughout the entire ACE2 sequence 95 at only 79% compared to the other species, but is 84% identical within the identified interacting 96 residues. In contrast, dogs are 83.3% identical across the entire sequence, but only 76% identical in 97 the interacting residues. A similar lower identity in binding site interacting residues is also seen for 98 rabbits (Table 1 ). The relative contribution of the interacting residues versus residues outside the RBD 99 binding site is currently not known, although it is expected that the interacting residues are far more 100 important to infection relative to the residues outside of the RBD binding site. 101

For variability analysis, the structural importance of protein regions, and even individual amino acid 102 residues, can be compared across multiple species. Such diversity analyses initially identified the 103 complementary determining regions within antibodies as important interacting domains with antigen by 104 Kabat and Wu(32, 33) , and more recently Shannon entropy evaluation has been employed to identify 105 conserved and diverse domains of multiple proteins through multiple sequence analysis(34,35). First, 106

we aligned the ACE2 sequences and determined their percent identities (Table 1 and Supplemental  107   Table 1 ). Then, we calculated Shannon entropy (SE) across the ACE2 sequences. Of note, ACE2 is 108 remarkably conserved across its sequence, with few residues exhibiting high variability ( Figure 2) . 109

Within the interacting residues, 21 of 25 are highly conserved, with SE values below 2. Significantly, no 110 residues had values above 3. The more variable residues are colored red and conserved residues blue 111 in Figure 2 . Five residues, N330, G352, D355, R357, and R393, are completely conserved, with SE 112 values of zero ( Figure 2 and Supplemental Table 1 ). Four of these are nearby residues with RBD, with 113 only R357 being a contact residue. The complete conservation of these residues suggests that they 114 play an important role in the protease function or structural integrity of ACE2. Residues with values 115 between 0 and 1 are K353, which is a contact residue, and L45, Y83, T324, which are nearby residues 116 (Supplemental Table 1 ).

Amino acids with SE values between 1-2 are K26, T27, D30, K31, E35, 117 D38, Q42, M82, and G354. The most diverse residues, with SE values over 2, are Q24, H34, L79, and 118 Q81. Of these, Q24 and H34 are contact residues, with H34 found in a central location in the ACE2-119 RBD interface (Figure 1 , B), and having by far the highest SE value at 2.88 . Others have analyzed the 120 evolution of ACE2 residues and have found positions 24 and 34 to be undergoing positive evolutionary 121 selection pressure (36), and suggested that these positions could play a role in predicting infectivity by 122 SARS-CoV-2 (36). 123

Since the interacting residues are likely most important for viral interaction with ACE2 on the host cell, 124

we evaluated the residues that differed between the various species' ACE2 and human ACE2 in this 125 region ( Figure 3 and Supplemental Figure 3 ). As mentioned, horseshoe bat (Rhinolophus sinicus) 126

shares only 19/25 interacting residues (76%) with human ACE2, and only 5/8 contact residues. 127 Specifically, D30E, H34T, Y41H, and M82N (contact residues), and T27M, E35K (nearby residues) are 128 mutated in horseshoe bat ACE2 relative to human ACE2 (Figure 3 Cows serve as a reservoir for bovine coronavirus (BCoV) a respiratory infection of cattle that is a 139 betacoronavirus (11,12) distantly related to SARS-CoV-2(7). Of considerable note, two BCoV-related 140 coronaviruses, HCoV-OC43 and HCoV-HKU1, have crossed the species barrier, with OC43 likely from 141 cows to humans to cause "common cold" respiratory disease in humans(37). Whereas BCoV, HCoV-142 OC43 and HCoV-HKU1 utilize 9-O-acetylated sialoglycans as cellular receptors(38), and SARS-CoV-2 143 utilizes ACE2, cows are a potential important species to evaluate for possible SARS-CoV-2 infection as 144 they are a known coronavirus reservoir and they could theoretically serve as host for SARS-CoV-2 and 145 other coronaviruses like BCoV, which could potentially provide a host for coronavirus recombination, 146 selection, and evolution. From a biochemical standpoint, cows have a somewhat more distantly related 147 ACE2 protein compared to many other vertebrates (78.8% compared to most other species which are 148 over 80%), however their binding site residues are more conserved (84%)( Table 1) . Therefore, it would 149 be useful to know whether the lower homology across the entire ACE2 sequence prohibits productive 150 ACE2/RBD interaction. To address this question, we expressed human and bovine ACE2 as antibody 151

Fc fusion proteins and compared their interaction with SARS-CoV-2 by enzyme linked immunosorbent 152 assay (ELISA) and further quantified their binding kinetics by surface plasmon resonance analysis 153 ( Figure 4 ). By ELISA, bovine ACE2 had an approximately ten-fold worse binding EC 50 (0.129 nM for 154 human ACE2 compared to 1.299 nM for bovine ACE2) (Figure 4 , A). This lower apparent affinity for 155 bovine ACE2 was confirmed by surface plasmon resonance analysis which showed a KD for bovine 156 ACE2 of 36.25 nM versus 7.5 nM for human ACE2 (Figure 4 , B and Supplementary Table 1 ). This 157 dissociation constant difference relates primarily to a faster off-rate for bovine ACE2 compared to 158 human ACE2 (Supplementary Figure 5 ). Of note, despite this lower KD for bovine ACE2, this affinity is 159 very similar to the KD for human ACE2 for the RBD of SARS-CoV-1. Interestingly, for both human and 160 bovine ACE2, the SPR data fit more consistently with a two-site model for interaction, suggesting that 161 the RBD may be multimerizing to produce avidity effects on the chip surface. A potential second site 162 would have ten-fold lower KD values (Supplemental Figure 5 ), which interestingly, are more in line with 163 the EC50 values of the ELISA (Figure 4 , A). Such interactions would be important to explore to 164 understand the details of interaction between coronavirus spike RBD and ACE2 on the cell surface. 165

These interactions would be important to inhibit by therapeutic agents, for example by monoclonal 166 antibodies targeting the virus. The avidity interaction may decrease the KD (increase the affinity) of 167 bovine ACE2 with spike RBD from 36 nM to 2.5 nM, and 7.5 nM to 0.4 nM for human ACE2, a 168 substantial enhancement of the interaction. Regardless of the mechanisms of interaction, it is clear that 169

bovine ACE2 still has high affinity towards SARS-CoV-2 RBD, albeit with five to ten fold worse binding 170 than human ACE2, but yet can still mediate infection of bovine cells (18). 171 172 Discussion 173

The recent COVID-19 pandemic has spread rapidly across the globe through human populations, but 174 additionally has also infected several animal species. While zoonotic in origin, it is still unclear which 175 species provided the reservoir for transfer to humans. Coronaviruses as a group have a very wide 176 range of host species, and have jumped the species barrier multiple times to humans(39,40). While 177 bats appear to be a host species for coronaviruses related to SARS-CoV and SARS-CoV-2, it is 178 possible that an as yet unidentified species serves as the reservoir for this virus. Additionally, it is clear 179 that SARS-CoV-2 can naturally infect other species, such as cats, dogs, and mink. In order to 180 understand the host range of SARS-CoV-2 as well as identify potential reservoirs for the virus it is 181 critically important to understand (i) details about the identity and binding properties between the virus 182 and its host cell receptor and possible co-receptors, (ii) other biological requirements needed for the 183 virus to replicate and transmit, for example host cell enzymes needed for viral processing, replication, 184 and assembly. For SARS-CoV-2, ACE2 appears to be the major receptor required for cell entry, so 185 understanding its interaction with ACE2 from humans as well as other species is important to enable 186 predictive methods for viral host range. Here we analyze ACE2 diversity across several species, and 187 specifically evaluate binding of SARS-CoV-2 RBD to bovine ACE2, finding key interacting residues to 188 be highly conserved. 189

Several studies have used sequence homology and/or structural modeling to attempt to predict CoV-2 RBD binding to various species' ACE2 in order to predict the virus host range (15, 36, (39) (40) (41) (42) (43) . In 191 an effort to predict species permissive to infection, Damas et.al. developed a five-tiered scoring scheme 192 based on percent identity of 410 vertebrate species, as well as specific structural features of CoV-1 or SARS-CoV-2 interactions with ACE2(36). They also focused on 25 amino acid residues at 194 the RBD binding interface, however their 25 residues differed from ours in that they included S16, N53, 195 N90, and N322, with the asparagines included as potential glycosylation sites that may impact RBD 196 binding. However, our approach was agnostic in choosing residues that were either (i) known contact 197 residues with the RBD, or (ii) within 5 angstroms of contact residues. Residues included in our analysis 198 which were not included in Damas et.al. were K26, Q81, and N352. Damas et. al. also note that the 199 host range of SARS-CoV-2 might be quite broad and suggest new species that should be evaluated for 200 animal models of virus infection, which notably include cows, which scored in their "medium" category 201 for predictive binding to RBD. Like our assessment of homology, they find that bats score very low in 202 predicted ACE2 binding. Interestingly, using sequence evolution and selection analysis, they identify 203 Q24 and H34 as positions undergoing positive selection and evolution. We identified these as amongst 204 the most variable residues by Shannon entropy analysis, and these also appear to be important 205 residues at the ACE2/RBD interface. As in our analysis, pangolins scored low in potential RBD binding 206 based on interacting residue homology, suggesting that SARS-CoV-2 may bind other pangolin 207 receptors, or have other mechanisms to interact with ACE2. As pangolins are thought to be a possible 208 intermediate host for SARS-CoV-2, much more biochemical and infectivity data with this controversial 209 species should be obtained. 210

In a different approach, Lam et.al. used structural modeling to predict the change in free energy, ∆∆G, 211 for 215 ACE2 sequences derived from different species(44). They correlated the ∆∆G with published 212 infectability information to provide a framework to predict which species may be susceptible to SARS-213

CoV-2 infection. Like other studies, their work suggests a broad range of mammal susceptibility, with 214 the exception of non-placental mammals. They similarly find that horseshoe bats have higher ∆∆G 215 values (i.e. lower affinity), calling into question their susceptibility and potential as a reservoir for SARS-216

CoV-2. 217

There have been several studies that have measured the K D between SARS-CoV-2 spike (or RBD) and 218 ACE2, with values ranging from 1.2 to 44 nM (23, 25, 29, 30) . In these studies, differences in the 219 experimental conditions, such as whether ACE2 or RBD was immobilized, and technique used such as 220 biolayer interferometry versus surface plasmon resonance, could account for differences in kinetic 221 values. In our study, we immobilized ACE2 and measured RBD interaction as the analyte using 222 (45) . Since the RBD exists in close proximity to two other 228 RBD subunits in the spike protein trimer, it is plausible that cooperative interactions between RBDs 229 exist during ACE2 interaction events. Such events could considerably enhance the interactions 230 providing avidity to enable infection. In this regard, ACE2 variants from species with mutations in the 231 RBD interface may still be permissive to infection because of the cooperative and enhanced avidity of 232 binding events. 233

One of the major challenges in developing predictive methods for viral infectivity is the limited amount 234 of both infection and RBD-ACE2 biochemical interaction data for most species. For example, only few 235 species have had documented infection in the real world, or even in well-controlled experimental 236 systems (15, 36, 40, 44) . In this regard, detailed biochemical analysis of multiple species' receptors 237 would provide valuable information that could be used in predictive modeling studies. With the 238 decreased costs of synthetic DNA and high-throughput screening approaches, such analyses could 239 potentially be accomplished rapidly. As a first step in this process, here we evaluate cow ACE2 for 240 binding SARS-CoV-2 RBD by both ELISA and surface plasmon resonance, a species with documented 241 experimental infection by SARS-CoV-2 and of significant agricultural importance that serves as a 242 reservoir for other betacoronaviruses, including at least one, HCoV-O43, that has jumped the species 243 barrier to become an endemic respiratory virus in humans. The affinity of bovine ACE2 is nearly ten-244 fold worse than human ACE2 in binding the coronavirus RBD, however binding is still in the mid-245 nanomolar range. Of potential importance is the possible cooperativity in binding that may occur 246 between ACE2 and RBD, which we observed in surface plasmon resonance analysis which suggested 247 a two-site model for binding. Interestingly, this data was consistent with ELISA binding data, 248 suggesting K D in the subnanomolar range for human, and low nanomolar range for bovine ACE2. 249

Further study into various species' viral receptors, including the recently discovered neuropilin-2 co-250 receptor for SARS-CoV-2 (46) were identified based on the co-crystal structure (27), and we additionally added 17 ACE2 residues 273 (nearby residues) that were within at least 5 angstroms of the RBD region and classified the full list as 274 "interaction residues". Thus, there were a total of 25 interaction residues, with 8 known RBD contact 275 residues and 17 nearby residues. Using a de novo python script, these residues were extracted into a 276 separate "sequence" and an additional, specific multiple sequence alignment was constructed. 277

The interaction residue multiple sequence alignments were investigated to give more focused insight 278 into which species would be at risk of infection, with the assumption that residues directly interacting 279 with the SARS-CoV-2 spike protein would be essential for infection. Structural analyses were 280 performed in Visual Molecular Dynamics (VMD, https://www.ks.uiuc.edu/Research/vmd/). 281

Human and bovine ACE2 proteins were produced as fusion proteins to human IgG1 Fc according to 283 our published methods for monoclonal antibody purification [2] [3] [4] . Briefly, 30 M HEK293 Freestyle cells 284

were transfected with 293fectin combined with 30 µg of pFuse-based vectors containing the human or 285 bovine ACE2 fused to the human Fc region. Cells were shaken at 37 °C for 4 days with 8% CO2. The 286 media was clarified by centrifugation at 4000 RPM for 5 minutes followed by filtration through a 0.22 µm 287 filter. The media was concentrated and buffer-exchanged into PBS using Amicon Ultra Centrifugal Filter 288 unit (MWCO = 10,000) (MilliporeSigma) at 4 °C. The concentrated media was then loaded onto a 289 protein A-sepharose column (Cytiva) pre-equilibrated with 20 mM sodium phosphate, pH 7.0, followed 290 by washing of the column with 10 column volumes of the same buffer and eluted twice with 1 column 291 volume of 0.1 M glycine-HCl, pH 2.7 into fractions containing 0.1 column volume of 1M Tris, pH 8. 292

Purified proteins were buffer-exchanged into PBS using Amicon Ultra Centrifugal Filter unit (MWCO = 293 10,000) (MilliporeSigma), quantified using 280 nM absorbance on a Nanodrop spectrophotometer 294 (Thermo Fisher Scientific), and resolved on an SDS-PAGE stained with InstantBlue Coomassie Protein 295 Stain (Abcam). 296

The SARS-CoV-2 RBD in plasmid NR-52309 (BEI Resources) was transfected and harvested as 297 described above, but purified using TALON cobalt metal affinity resin (Takara Bio) following the 298 manufacturer's protocol, except that 50 mM, 100 mM, 200 mM and 300 mM imidazole gradient elution 299 fractions (1 column volume of each) were collected. Each elution fraction was resolved on an SDS-300 PAGE gel stained with InstantBlue Coomassie Protein Stain (Abcam), and fractions containing a single 301 RBD band were pooled, buffer-exchanged into PBS and quantified as described above. Purified 302 protein was resolved on SDS-PAGE with Coomassie (Gel-code blue) staining. 303 D355, R393). Throughout the manuscript we refer to these as "contact residues" (purple) and "nearby 344 residues" (cyan), and together as "interacting residues". (B) Top down view of ACE2 in space filling 345 mode, with residues color-coded as in (A). G352 is not visible in this orientation. (C) Boxshade 346 alignment of only the contact residues and nearby amino acid residues from (B) for multiple species. 347

Contact residues are indicated with a cyan bar on top of the sequence, and nearby residues with a 348 purple bar. 349 entropy values projected onto the human ACE2 structure as a heat map from blue (low) to red (high). 354

(Right) View of the RBD-interacting surface as in Figure 1 , with contact and nearby residues labeled. 355

Blue residues are very highly conserved. 356 357 Figure 3 . Variable residues at the ACE2-RBD interface of individual species. With human ACE2 358 as a reference, the variant interacting residues for each species are colored red, and conserved 359 residues colored cyan. For the remainder of the ACE2 protein, conserved residues are white and 360 variable residues light red. Certain residues like M82, Q24, D30, and H34 are often mutated relative to 361 human. D30 is conservatively changed to glutamate, however more non-conservative changes can be 362 seen for H34, for example. More species have been analyzed and are shown in Supplemental Figure  363 3. 364 The following reagent was produced under HHSN272201400008C and obtained through BEI 382

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