METHODOLOGY

The aim of this research is to look at the possible effect of missense mutation at ACE2 receptor with regards to its
binding efficiency with SARS-CoV 2 spike glycoprotein. By using Cao, et. Al (2020) research about different ACE2 variants
in different populations, the researchers chose single nucleotide polymorphisms (SNPs) found in the exons of ACE2 gene.
Specifically, SNPs found at the peptidase domain of the ACE2 protein are given much more focus.

The crystal structure used in this research was obtained from the Protein data base. There is already an existing model
of the actual binding of the spike glycoprotein of the SARS CoV-2 to the receptor binding domain of the ACE2 receptor.

Figure 123. The model structure of ACE2 receptor interacting with the spike glycoprotein of SARS-CoV-2 obtained from
the protein data bank.

The mutation was done by simply changing the amino acid residue in the protease domain of the ACE2 receptor with the
research of Cao, et. al used as the reference. The different mutations are listed in table XYZ. Chimera 1.14 was the
software used in inducing the mutation in the normal ACE2 variant. After changing the amino acid residue, possible
clashes between different molecules are determined and the resulting protein structure is was minimized in order to
optimize the variant.

TABLE XYZ. List of the different ACE2 variants and their type of missense mutation.
Amino acid position Amino acid in the normal variant Variant amino acid
21 Isoleucine Threonine
26 Lycine Arginine
33 Asparagine Aspartate
38 Aspartate Glutamate
75 Glycine Valine
149 Asparagine Serine
159 Asparagine Serine
194 Valine Alanine
383 Methionine Isoleucine
478 Isoleucine Valine
592 Phenylalanine Leucine
After minimizing the structure of the resulting variant, the distance between the interacting molecules between the
protein binding domain of ACE2 and the spike glycoprotein of the SARS CoV 2 were measured using the software
PyMOL. The results, together with their mean are seen in Table ABC.

RESULTS

Table ABC. The distance obtained between interacting molecules in ACE2 and SARS CoV2.
DISTANCE (Angstroms)
N-TERMINUS MIDDLE C-
VARIANT MEAN
TERMINU
S
1 2 3 4 5 1 2 1
NORMAL 4.9 3.4 5.1 5.7 5.8 3.8 2.9 9.7 5.1625
ASN33ASP 4.9 3.4 5.1 5.7 3.7 3.8 2.9 9.7 4.9
ASN149SER 4.9 3.4 5.1 5.7 3.7 3.8 2.9 9.7 4.9
ASN 159SER 4.9 3.4 5.1 5.7 5.8 3.8 2.9 9.2 5.1
ASP38GLU 4.6 3.4 5.1 5.7 5.8 3.8 2.9 9.7 5.125
GLY75VAL 4.9 3.4 5.1 5.7 5.8 3.8 2.9 9.7 5.1625
ILE21THR 4.9 3.4 5.1 5.7 5.8 3.8 2.9 9.7 5.1625
ILE478VAL 4.9 3.4 5.1 5.7 5.8 3.8 2.9 9.7 5.1625
LYS26ARG 4.9 3.4 5.1 5.7 5.8 3.8 2.9 9.7 5.1625
MET383ILE 4.9 3.4 5.1 5.7 5.8 3.8 2.9 9.7 5.1625
PHE592LEU 4.9 3.4 5.1 5.7 5.8 3.8 2.9 9.7 5.1625
VAL194ALA 4.9 3.4 5.1 5.7 5.8 3.8 2.9 9.7 5.1625

STATISTICAL ANALYSIS
DISCUSSION

The Severe acute respiratory syndrome Coronavirus 2 (SARS CoV-2) is the cause of the coronavirus disease 2019
(COVID19). It is a virus first identified in Wuhan, China that spread and caused a global pandemic. Infection of this virus
varies from mild symptoms like fever, cough, fatigue and shortness of breath to severe conditions like acute respiratory
distress syndrome (ARDS), multi-organ failure, septic shock, and blood clots.

SARS-CoV-2 specifically binds to ACE2 receptor in order for it to enter the cell and replicate. By using its spike
glycoprotein, it interacts with the protein binding domain found at the protease domain of ACE2 and this interaction
allows the virus to be endocytosed by the cell. ACE2 receptors are most abundant in Type II alveolar cells of the lung
making it the most affected organ.

The full length ACE2 receptor consists if an N-terminal protease domain (PD), and a C-terminal collectrin-like domain
(CLD) which consists of a small extracellular domain, a long linker, and ends with a single transmembrane helix. The
structure and domains of the ACE2 receptor is shown in figure 456 (Yan et.al, 2020). The PD of the ACE2 receptor is
responsible for cleaving Angiotensin I to produce Angiotensin (1-9).

Figure 456. ACE2 structure model showing its different domains—PD and CLD (Yan et.al, 2020). .

The PD of ACE2 accommodates one Receptor binding domain (RBD) from the SARS-CoV-2 virus. The contact between
the RBD of the virus and the α1helix of ACE2 PD is divided into three clusters—the N- and C- termini and the middle
segment between the two. At the N-terminus of the α1helix, the RBD of SARS-CoV-2 has its amino acids Gln 498, Thr500,
and Asn501 interact with the Tyr41, Gln42, Lys353 and Arg357 of ACE2. In the middle section, Lys417 and Tyr453 of the RBD
interact with Asp30 and His34 of ACE2 respectively. Lastly, at the C terminus, amino acids Gln 474 of the RBD interacts with
Gln24 of ACE2 and through van der Waals forces, the Met 42 of ACE2 interacts with Phe486 of the RBD (Yan et.al, 2020).
 Figure 678. Interactions between SARS-CoV-2 RBD and α1helix of the PD of ACE2 (Yan et.al, 2020).

With the premise that a missense mutation may change the structure of a protein, an assumption that there is a
possibility that one of the ACE2 variants (i.e. SNPs that caused missense mutations in ACE2 receptor) affects the
predisposition of certain population to SARS-CoV-2 infection was made since the amino acid-change may affect its
binding efficiency. Using the mean distance between the two interacting portion of the ACE2 receptor and SARS-CoV-2
as the parameter, we determined which among the ACE2 variants (and the population which have it) are more or less
prone with Covid-19.

Looking at table ABC, variations that occurred at amino acid positions 21, 26, 75, 194, 383, 478, and 592 did not affect
the binding efficiency of the spike glycoprotein since the mean distance between the interacting surfaces of the virus
and the ACE2 receptor did not change. Possible explanation for this is that the side chains of the changed amino acids
have not altered the electrochemical interactions between other molecules allowing the variant protein to still have
their original structure. Another possible explanation is that the SNP that the variant ACE2 receptor have is not found or
is far from the α1helix of the ACE2 receptor thus, it gives no or little effect on the binding of the virus to the receptor.

On the other hand, ACE2 variants with SNPs at amino acid positions 33, 38, 149, and 159 have a mean distance lower
than what was found in the normal variant. This may mean that the efficiency of binding of the virus with these ACE2
receptor variants is higher as compared to the normal and other variants. However, the result of the statistical analysis
says otherwise. Upon subjecting the results to 1-way ANOVA, the means did not have significant differences (p-value =
1.000 > 0.05). This may indicate that even though there are changes in the structure of the protein due to these SNPs,
these interindividual alterations in the amino acid sequence are not significant enough to greatly affect the binding
efficiency of the virus to these variant ACE2 receptors. With this result, we cannot say that the population containing
these ACE2 variants are more susceptible the SARS-CoV2 infection.

CONCLUSION

With SARS-CoV2 causing pandemic, different researches about its origin, pathophysiology and countermeasures against
it are being made. Since it is easily transmitted, it poses a great danger since it can affect a large area in a short period of
time and the effects it produce when one acquires it can lead to death if no actions are made immediately. This research
aims to see who among the population with different ACE2 receptor variants are more predisposed to the infection thus
allowing people to create better plans in order to handle and control this pandemic.

The mean distance between the interacting surfaces of the ACE2 receptor and the spike glycoprotein of the virus were
obtained in different ACE2 receptor variants that have SNPs found in their coding regions. This aims to evaluate the
binding efficiency of the virus with its receptor by looking how the SNPs affect the protein structure thus also ultimately
affecting how the virus binds to its receptor. Using 1-way ANOVA, the p-value of 1.000 was obtained. This is greater than
0.05 thus indicating that the differences in the means are NOT SIGNIFICANT enough to say that the SNPs found in the
coding region of the ACE2 protein make the certain population having these variants more susceptible to virus infection.

The result may indicate that the SNPs found in the coding regions of the ACE2 gene do not really affect the infectivity of
the SARS-CoV2 virus. These SNPs in the coding region that caused missense mutations may have altered amino acids
that do not greatly affect the intermolecular interactions within the protein thus resulting to no change in its structure
especially at the regions that interact with the virus. With this, we cannot say that these mutations made the population
more or less predisposed with the SARS-CoV 2 infection. However, these changes may affect its physiological function
and further studies must be done in order to confirm which among these variants, if any, influence how Angiotensin I is
metabolized by this protein.

On the other hand, SNPs found on the noncoding regions may have an effect since those SNPs may affect the gene’s
expression. For instance, a SNP found in the noncoding region may allow enhancers and other transcription factors to
efficiently bind to the gene and increase its expression. If there is an increase in expression, there will also be an
increased presence of ACE2 receptor in the plasma membrane of cells thus allowing more virus to infect it. The opposite
may also be true regarding this. A SNP can also decrease the binding of different proteins and transcription factors
decreasing the amount of ACE2 receptor found on the cell’s surface thus reducing its predisposition to the virus
infection. However, these are merely claims and these need to be looked further in order for it to be verified.

Bibliography
Yan, R., Zhang, Y., Li, Y., Xia, L., Guo, Y., & Zhou, Q. (2020). Structural basis for the recognition of SARS-CoV-2 by full
length human ACE2. Science, 1-5.

Cao, Y., Li, L., Feng, Z., Wan, S., Huang, P., Sun, X., … Wang, W. (2020). Comparative genetic analysis of the novel
coronavirus (2019-nCoV/SARS-CoV-2) receptor ACE2 in different populations. Cell Discovery, 6(1). doi:10.1038/s41421-
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