key: cord-0885842-m0w0fl2u
authors: Hussain, Mushtaq; Jabeen, Nusrat; Raza, Fozia; Shabbir, Sanya; Baig, Ayesha A.; Amanullah, Anusha; Aziz, Basma
title: Structural variations in human ACE2 may influence its binding with SARS‐CoV‐2 spike protein
date: 2020-04-15
journal: J Med Virol
DOI: 10.1002/jmv.25832
sha: 343736ab230410afe85bb957623564ada151d316
doc_id: 885842
cord_uid: m0w0fl2u

The recent pandemic of COVID‐19, caused by SARS‐CoV‐2, is unarguably the most fearsome compared with the earlier outbreaks caused by other coronaviruses, SARS‐CoV and MERS‐CoV. Human ACE2 is now established as a receptor for the SARS‐CoV‐2 spike protein. Where variations in the viral spike protein, in turn, lead to the cross‐species transmission of the virus, genetic variations in the host receptor ACE2 may also contribute to the susceptibility and/or resistance against the viral infection. This study aims to explore the binding of the proteins encoded by different human ACE2 allelic variants with SARS‐CoV‐2 spike protein. Briefly, coding variants of ACE2 corresponding to the reported binding sites for its attachment with coronavirus spike protein were selected and molecular models of these variants were constructed by homology modeling. The models were then superimposed over the native ACE2 and ACE2‐spike protein complex, to observe structural changes in the ACE2 variants and their intermolecular interactions with SARS‐CoV‐2 spike protein, respectively. Despite strong overall structural similarities, the spatial orientation of the key interacting residues varies in the ACE2 variants compared with the wild‐type molecule. Most ACE2 variants showed a similar binding affinity for SARS‐CoV‐2 spike protein as observed in the complex structure of wild‐type ACE2 and SARS‐CoV‐2 spike protein. However, ACE2 alleles, rs73635825 (S19P) and rs143936283 (E329G) showed noticeable variations in their intermolecular interactions with the viral spike protein. In summary, our data provide a structural basis of potential resistance against SARS‐CoV‐2 infection driven by ACE2 allelic variants.

whereas MERS-CoV spike protein attaches with dipeptidyl peptidase 4 (DPP4). 6, 7 Recently, several structures of SARS-CoV-2 spike protein complexed with human ACE2 have been resolved (PDBids: 6LZG, 6VW1, 6M17), highlighting the critical residues involved in the intermolecular interactions between the viral spike protein and its receptor. 7 As the virus has been transmitted in humans from bats and/ or other intermediate hosts, considerable work has been conducted to explore the sequence and structural variations in the spike proteins and animal host ACE2 receptors. [8] [9] [10] It has been shown that variations in both viral spike protein and host ACE2 sequences may act as a barrier for viral infection across species. 2, 8, 9 This, in turn, raises two interesting possibilities: first, do the natural genetic polymorphism in human ACE2 gene and/or protein influence their attachment with SARS-CoV-2 spike protein? Second, are these genetic variants of ACE2 benign or could it be considered as an evolutionary trade-off where a variation in a trait may bring both advantage and disadvantage to the fitness of an organism? This study aims to explore both these possibilities by a battery of sequence and structure analysis tools. The findings provide interesting insights into the potential relationship between natural genetic variants in human ACE2 and susceptibility and/or resistance against COVID-19 infection. and human ACE2 (Q9BYF1) were retrieved from NCBI and UniProt, respectively. Structures of both the proteins were identified by PDB-BLAST and retrieved from RCSB protein data bank. 11 Data for genetic variants and allele frequencies were obtained from Ensembl Genome Browser 12 and gnomAD. 13 Appropriate filters were employed to limit the data to only the coding region variants of ACE2 gene. Coding variants of ACE2 corresponding to the critical binding sites between ACE2-SARS-CoV 14 and ACE2-SARS-CoV-2 (PDBid: 6LZG), and those reported to show impaired binding with coronavirus spike protein in the in vitro mutation analysis 15 were selected for further investigations (Table S1 ).

The effect of amino acid substitution on protein (ACE2) stability was predicted at 25°C and 37°C using I-Mutant2.0. 16 The program predicts the change in the free energy (ΔΔG) in the sequence variants compared with the wild type. An increase or decrease in protein stability is indicated by negative or positive ΔΔG values, respectively. The functional impact of all selected allelic variants of ACE2 was predicted using sorting intolerant from tolerant (SIFT), 17 PolyPhen-2, 18 combined annotation-dependent depletion (CADD) 19 and rare exome variant ensemble learner (REVEL). 20 

A total of 17 ACE2 coding region allelic variants were identified that correspond to the critical interaction points between ACE2 and receptor-binding domain (RBD) of coronavirus spike protein.

Structural models of these variants were generated using atomic coordinates of PDBid: 2AJFA (wild-type ACE2) as a template by 

SARS-CoV-2 spike glycoprotein is a 1273 amino-acid-long structural protein located on the outer envelope of the virus. The protein has two main functional subunits: a long N-terminal S1 subunit and a relatively short C-terminal S2 subunit. A 200 amino-acid-long RBD is stationed within the S1 subunit of the spike protein that is mainly involved in its interaction with ACE2 receptor. 6,7,10 Human ACE2 is an 805 amino-acid-long protein with two functional domains:

N-terminal peptidase M2 domain and C-terminal collectrin domain.

Partial structure of the ACE2 has been resolved (PDBid: 1R42) comprising peptidase M2 domain. Structurally, the peptidase domain has two catalytic subdomains with an active site sandwiched in between the two subdomains. 24 Cocrystal structures of SARS-CoV-2 spike protein complexed with human ACE2 (PDBid: 6LZG), SARS-CoV with human ACE2, 14 and in vitro mutation analysis of human ACE2 15 have identified the critical residues that underpin the interaction between the RBD of viral spike protein and ACE2 peptidase domain. In this study, among the 345 and 242 natural ACE2 coding variants identified from Ensembl Genome Browser and gnomAD, respectively, 17 were found at positions that have shown to be important for the binding of ACE2 with the viral spike protein (Table S1 ). Allele frequencies of these variants range from 5.45E−6 (rs1299103394) to 3.88E−3 (rs4646116). To predict the potential pathological consequences rendered by ACE2 alleles, changes in the free energy of ACE2 variants were estimated. The results showed that amino acid substitutions in only three alleles rs73635825 (S19P), rs1299103394

(K26E), and rs766996587 (M82I) may adversely affect the stability of the encoded protein compared with the wild type (Table 1) . However, to date, no pathogenic consequences have been reported in relation to these variants in humans. Moreover, except for rs73635825 (S19P), which was predicted to be damaging by only PolyPhen-2, different prediction tools designed to assess the functional impact of the mutations and/or single-nucleotide polymorphisms did not reveal any ensuing pathogenicity rendered by these variants (Table 1) .

Conversely, rs961360700 (D355N) and rs762890235 (P389H) were predicted to be detrimental by SIFT and Polyphen-2, whereas rs1396769231 (M383T) was predicted to be damaging by SIFT, Polyphen-2, and REVEL prediction tools. Nevertheless, no report of any functional impact was found in the literature for these allelic variants of ACE2 as well.

To explore the structural changes in the protein encoded by Figure 2C ). Many of these interactions were also reported in a previously resolved structure of SARS-CoV spike protein-ACE2 complex (PDBid: 2AJF). 14 A representation of the comparison of these interactions for different ACE2 alleles is shown in Figure 2D .

Briefly, the most conserved intermolecular interactions (hydrogen bonds) between different ACE2 variants and SARS-CoV-2 spike protein were found between Y41, H34, Y83, and K353 of ACE2 spike protein. 15 Therefore it is likely that rs73635825 (S19P) and (S19P) and rs143936283 (E329G). Therefore, the findings of this investigation provide clues to screen frequencies of the candidate alleles in different populations to predict the prognosis of COVID-19.

http://orcid.org/0000-0001-8862-179X

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