Research Article - (2020) Volume 4, Issue 2
Received: 25-Aug-2020
Published:
29-Sep-2020
, DOI: 10.37421/vcrh.2020.4.116
Citation: Batista Joao Junior. "Interaction Energies of the Human
ACE2 Molecular Recognition by SARS-CoV-2."Virol Curr Res 4 (2020): 116.
Copyright: © 2020 Batista Júnior J. This is an open-access article distributed under the terms of the creative commons attribution license which permits
unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.
The Coronaviridae family of viruses includes hundreds of viruses common in many different animal species and humans. Seven coronaviruses (CoVs) are known to cause disease in humans. Four of them show low pathogenicity and are endemic in humans and the other three CoV are particularly dangerous and highly pathogenic viruses, which underwent genetic changes rendering them able to jump the species barriers from animal host to humans and also to spread efficiently among humans. SARS-CoV-2 is the seventh coronavirus known to infect humans. The S protein mediates attachment and viral and host cell membrane fusion. The receptor-binding domains (RBDs) are regions in S protein responsible for receptor recognition. Human angiotensin-converting enzyme 2 (ACE2) is recognized by HCoV-NL63, SARS-CoV and SARS-CoV-2 as their functional receptor.
Interaction energy analysis were performed to unveil how precisely SARS-CoV-2 interacts with ACE2 by identifying which amino acid residues are responsible for the interactions across S protein-ACE2 interfaces and how they contribute to the strength, stability and specificity of S protein interactions.
Interaction energies acting on molecular recognition of ACE2 by HCoV-NL63, SARS-CoV and SARS-CoV-2 conduced to a naturally evolved RBD with different combinations of amino acids, providing SARS-CoV-2 binding interface more interacting residue pairs, more hydrogen bonds, increased number of residues engaged in hydrogen bonding, allowing for better distribution of hydrogen bond per residue in interface than SARS-CoV or HCoV-NL63, includes salt bridge, and adds new van der Waals contacts into the network.
Residues across the SARS-CoV and SARS-CoV -2 homologous sequences have been chosen to be remarkably evolutionary conserved in the regions mediating binding of these viruses because of their dominant hydrogen bonding contribution to binding stability to ACE2. SARS-CoV-2 achieves higher binding affinity than SARS-CoV and HCoV-NL63 to human ACE2 molecular recognition primarily by combining its richer interaction network and higher binding stability.
This study presents a comprehensive and quantitative analysis of interaction energies of the human ACE2 molecular recognition by CoVs that may contribute to further understand the higher infectivity and transmissibility of SARS-CoV-2 compared to SARS-CoV and HCoV-NL63, furthermore, this could help explain why SARS-CoV-2 has an enhanced ability for pathogenicity.
Interaction energy • SARS-CoV-2 • Spike Glycoprotein • ACE2 • Molecular Recognition
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes an infectious disease named coronavirus disease 2019 (COVID-19). Since December in late 2019, when SARS-CoV-2 first made an appearance as a novel emerging coronavirus in Wuhan, China, it is infecting people and spreading easily, silently and rapidly from person-to-person worldwide. On March 11, 2020, the COVID-19 outbreak was characterized as a pandemic by the World Health Organization (WHO).
The Coronaviridae family of viruses includes hundreds of viruses, which are common in many different animal species, including wild animals (bats, civets, raccoons, pangolins) [1,2], domestic and peridomestic animals (cats, cattle, horses, pigs, goats, camels) [3], and humans [4,5]. This virus family consists of two subfamilies, Coronavirinae and Torovirinae (members of this subfamily are known to not cause human infection). The subfamily Coronavirinae comprises four genera called Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus [6].
Seven coronaviruses (CoV) are known to cause disease in humans. Four of them show low pathogenicity and are endemic in humans: human coronavirus-229E (HCoV-229E) and human coronavirus-NL63 (HCoVNL63), both from genus Alphacoronavirus (α-CoV); and human coronavirus- OC43 (HCoV-OC43) and human coronavirus- HKU1 (HCoV-HKU1), from genus Betacoronavirus (β-CoVs). Those CoV are an important cause of upper respiratory tract infections and have most frequently been associated with mild symptoms as those observed in the common cold [7]. The other three CoV are particularly dangerous and highly pathogenic viruses and all belonging to genus β-CoVs, included in the species Severe acute respiratory syndrome-related coronavirus (SARS-related CoV) [6], which underwent genetic changes, through mutation and/or recombination, rendering them able to jump the species barriers from animal host to humans and also to spread efficiently among humans [8,9] and cause much more severe respiratory infections, sometimes fatal, such as the outbreaks observed early in this century when severe acute respiratory syndrome coronavirus (SARS-CoV) emerged, in 2002, causing severe acute respiratory syndrome (SARS); Middle East respiratory syndrome coronavirus (MERS-CoV), in 2012, causing Middle East respiratory syndrome (MERS), and the current SARS-CoV-2, the seventh coronavirus known to infect humans, causing COVID-19 pandemic. All three SARS-related CoV posing a severe threat to public health.
The CoVs have a RNA genome, a positive sense, single-stranded genome, ranging from 26 to 32 kilobases (kb) in length, the largest genomes for RNA viruses, which encompasses a region encoding an RNA- dependent RNA polymerase, a region with coding sequences of genes which encode each one of the four main structural proteins required to produce a structurally complete viral particle: the spike (S) protein, envelope (E) protein, membrane (M) protein, the nucleocapsid(N), and a region representing several nonstructural proteins [10,11]. Some CoVs species may also possess a gene encoding the structural protein hemagglutininesterase (HE) [12].
Since CoVs have their genomic material surrounded by a lipid bilayer membrane and that genomic material needs to be transported through the barriers imposed by the host cell membranes, the S protein protruding from the viral surface mediates cell attachment and membrane fusion processes between the viral and target cell membranes. The S protein is a transmembrane glycoprotein that forms homotrimers. Each monomeric unit of S protein basically consists of three segments: an ectodomain, a transmembrane anchor and a short intracellular tail. The ectodomain comprises two functional subunits (S1 subunit and S2 subunit) used for invading host cells. S1 subunit is responsible for binding receptors and S2 subunit that contains the fusion machinery is responsible for viral and cellular membrane fusion. The S1 subunit N-terminal moiety comprises domain A. The S1 subunit C-terminal folds as three spatially distinct β-rich domains, termed domain B, C and D [13].
The regions responsible for receptor recognition in S protein are only found in domains A or B within S1 subunit, the receptor-binding domains (RBDs), and domain A or B is used in receptor recognition or attachment process specifically according to CoV species and their receptor specificities. A distinct location of S1 subunit domain A of HCoV-OC43 and HCoV-HKU1, both β-CoVs, mediates the binding of these viruses to the receptor 9-O-acetyl-sialic acid (9-O-Ac-Sia), which is terminally linked to oligosaccharides decorating glycoproteins and gangliosides, at the host cell surface [14]. HCoV-229E, an α-CoV, requires the zinc metalloprotease human aminopeptidase N as a receptor for entry into target cells and uses three receptor-binding loops of RBD present in S1 subunit domain B to bind aminopeptidase N [15,16]. MERS-CoV, a β-CoV, recognizes dipeptidyl peptidase 4 (DPP4) as its functional receptor by binding via its S1 subunit domain B. While MERS-CoV S1 subunit domain A selectively binds to sialoglycoconjugates on cell-surface which can serve as an attachment factor for support biding of S1 subunit domain B [17-19].
HCoV-NL63, a prevalent human respiratory virus, uses S1 subunit domain B, its RBD, to recognize angiotensin-converting enzyme 2 (ACE2) as its receptor for infection of target cells (Figure 1A). HCoV-NL63 is the only α-CoV known to use ACE2 as its receptor [20,21]. SARS-CoV and SARS- CoV-2, both β-CoV, also recognize host receptor ACE2 as its functional receptor and uses their S1 subunit domain B, their RBD, to attache the virion directly with ACE2 (Figures 1B and 1C) [22-24].
Figure 1. Recognition of human ACE2 by (A) HCoV-NL63, (B) SARS-CoV, and (C) SARS-CoV-2 RBDs. Surface representations of human ACE2 (in gray), HCoV-NL63 RBD (in yellow), SARS-CoV RBD (in green) and SARS-CoV-2 RBD (in red).
S protein is cleaved at the boundary between the S1 and S2 subunits in many CoVs, and those subunits remain non-covalently bound in the prefusion conformation [25]. The S protein of SARS-CoV-2 has a functional polybasic (furin) cleavage site at the S1–S2 boundary through the insertion of 12 nucleotides, which additionally led to the predicted acquisition of three O-linked glycans around the site [26]. After binding of RBD in S1 subunit of S protein on the virion to the ACE2 receptor on the target cell, the heptad repeat 1 (HR1) and heptad repeat 2 (HR2) domains in its S2 subunit of S protein interact with each other to form a six-helix bundle (6-HB) fusion core, bringing viral and cellular membranes into close proximity for fusion and infection [27].
In addition to being a cellular entry receptor for HCoV-NL63, SARS- CoV and SARS-CoV-2, ACE2 has its own unique functions. Human ACE2 is a glycoprotein, type 1 transmembrane metallopeptidase, expressed and active in most tissues, with remarkable expression observed on lung alveolar epithelial cells, enterocytes of the small intestine, and vascular endothelial cells and arterial smooth muscle cells [28]. ACE2 has an ectodomain containing its single zinccoordinating catalytic site on the cell surface. It functions as a carboxypeptidase and acts as regulatory components of the renin-angiotensin system (RAS), one of the most important hormonal systems in the physiological regulation of blood pressure and fluid balance. ACE2 hydrolyzes the C-terminal dipeptide of Angiotensin II (Ang II), a very powerful vasoconstrictor and the main active peptide of RAS, to convert it into Angiotensin 1-7 (Ang 1–7), a vasodilator. By regulating local levels of Ang II and Ang 1–7, in the cardiovascular system in particular, ACE2 has the importance in maintaining the balance of the RAS activation.
According to various studies, CoVs have existed early in the natural environments [29-31] and they have been present since 1966 in the human history [4]. CoVs have thus had plenty of time to adapt to their environments and to have given rise to numerous versions gaining ability to evolve to new restricted host, where there is less competition from other virus or life-forms.
In this study we unveil how precisely SARS-CoV-2 interacts with its functional host receptor by identifying which amino acid residues are responsible for the interactions across S protein-ACE2 interfaces and by detecting specific atoms from those amino acids and how they contribute to the strength, stability and specificity of S protein interactions.
To descript the atom-atom interactions across the interfaces of the S protein-ACE2 molecular complex, we selected from Protein Data Bank (PDB) experimental crystal structure for each CoV S protein RBD structure in complex with the human ACE2 receptor (for HCoV-NL63 PDB ID Code 3KBH, resolution 3.31 Å, [21]; for SARS-CoV PDB ID Code 2AJF, resolution 2.90 Å, [23]; and for SARS-CoV-2 PDB ID Code 6M0J, resolution 2.45 Å, [24]). We isolated each chain composing the molecular complex found in the crystal structures, removed water, ions, and all carbohydrates molecules bound to structure. After that, hydrogen atoms were added to the chains, followed by charges addition using AMBER force field. Next we performed interaction energy calculations [32] using parameters derived from AMBER parm99 molecular mechanical force fields for organic and biological molecules [33], in a solvent environment, to identify the key amino acid residues within CoV S protein RBD-ACE2 interfaces, with a maximum distance threshold of 4.00 Å, which are significantly contributing to the stability of that interaction.
The interatomic contact surface and interface areas were determined by calculating the S protein RBD and ACE2 complexed surfaces, the S protein RBD and ACE2 uncomplexed surfaces, and the buried surfaces for each unit in the complex [34].
Multiple sequence alignment of CoVs S protein sequences were computed using a progressive alignment construction method [35] for identifying residue conservation or residue changes in all sequences of the S protein RBDs.
Receptor recognition represents an important function in the process of virus adaptation to new hosts upon cross-species transmission of distinct viruses. An evolutionary and natural selected recognition mode can lead to new dominant genotypes. Since viral-receptor recognition relies on interfacial interaction energies, one can make the simplifying assumption that a dominant viral genotype is intrinsically linked to the interaction energies of the receptor recognition.
Sugar receptors have been serving to CoVs attach and entry to host cells for a long evolutionary time. Since protein receptors in general have advantages over sugar receptors by providing higher affinity interactions for viral attachment, natural selection events and evolution allowed CoVs to search for high-affinity protein receptors.
SARS-CoV-2 and SARS-CoV share high amino acid sequence conservation between their RBDs and low sequence conservation when compared to RBD of HCoV-NL63. Also, there is high structural similarity for SARS-CoV-2 and SARS-CoV RBDs, and no structural similarities with HCoV-NL63 RBD is observed. The S1 subunit of NL63-CoV, SARS-CoV, and SARS-CoV-2 may have undergone divergent evolution from a common ancestor into different structures and then convergent evolution to structures sharing ACE2-binding topologies. The amino acid interactions at the binding interface of HCoV-NL63, SARS-CoV and SARS-CoV-2 have progressively evolved in search of a stable binding network of residue–residue contacts to the human receptor ACE2. SARS- CoV-2 is a result of evolutionary optimized binding mode to the human receptor ACE2.
The interatomic interactions across the interfaces of the CoVs RBD- ACE2 molecular complexes show different combinations of amino acids. Residues across the SARS-CoV and SARS-CoV-2 homologous sequences have been chosen to be remarkably evolutionary conserved in the RBMs of this virus because of their dominant hydrogen bonding contribution to binding stability to ACE2 upon complex formation.
Furthermore, some residue mutations add new hydrogen bonds across the SARS-CoV-2-ACE2 interface and engage more residues in hydrogen bonding network enabling SARS-CoV-2 to have a more favorable hydrogen-bonding arrangement in the interface than SARS-CoV or HCoV-NL63, which contributes to enhance SARS-CoV-2-ACE2 complex complementarity and helps SARS-CoV-2 to achieve more specificity to ACE2 molecular recognition. Other mutations add new van der Waals interactions in the interface because they extend the number of residue contact pairs in ACE2, allowing them to enhance the number of new interatomic contacts, which leads to richer packing and considerable gain in van der Waals contribution to the binding stability. SARSCoV- 2 achieves higher binding affinity than SARS-CoV and HCoVNL63 to human receptor ACE2 not simply because of its enhanced number of interface interactions, but primarily by combining its interface interaction network optimization and the higher binding stability given by its RBD optimized interaction energies in human ACE2 molecular recognition.
Future research on this topic should yield significant new knowledge, however in the present context of receptor recognition mechanism those optimized interaction energies, higher binding stability, higher binding affinity would enable SARS-CoV-2, upon ACE2 binding, to initiate receptor-mediated signaling pathway resulting in its internalization into cell and in triggering a series of molecular and cellular mechanisms through which other signals are integrated during a productive infection causing a different disease outcome.
This comprehensive and quantitative analysis of interaction energies of the human ACE2 molecular recognition by CoVs may contribute to further understand the higher infectivity and transmissibility of SARS-CoV- 2 compared to SARS-CoV and HCoV-NL63, furthermore, this could help explain why SARS-CoV-2 has an enhanced ability for pathogenicity.
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