key: cord-0746276-a7xjnoha
authors: Bashir, Arif
title: Furin-cleavage site is present in an antiparallel β-strand in SARS-CoV2 Spike protein
date: 2022-02-18
journal: bioRxiv
DOI: 10.1101/2022.02.18.481028
sha: c93767566f12989b09a34526846f250434fe0252
doc_id: 746276
cord_uid: a7xjnoha

SARS-CoV2 spike (S) protein has been well recognized for its ability to bind with the angiotensin-converting enzyme (ACE2) receptor on human and other model lung epithelial cells. The furin cleavage-site (CS) present between the S1/S2 junction in SARS-CoV2 S protein is critical to drive the fusion between the SARS-CoV2 main body with the host cell. The available S protein structure lacks a stretch of amino acid including the furin CS as well. The majority of investigators have reported the presence of a loop harboring this patch. We are for the first time reporting this patch comprises of 14 amino acid residues (677QTNSPRRARSVASQ689) that form an antiparallel β-sheet comprising of PRRAR furin polybasic CS. We anticipate that this β-sheet is used as a scaffold by proteases to act on furin-CS in SARS-CoV2 S protein. Additionally, we studied the interaction of modeled SARS-CoV2 S protein with transmembrane protease, serine 2 (TMPRSS2), and furin proteases that accentuated that these proteases use furin CS sequence (PRRAR) located in β-sheet of our modeled SARS-CoV2 S protein to cleave the SARS-CoV2 S protein at S1/S2 junction.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)

SARS-CoV2 entry into host cells is facilitated by spike glycoprotein present on its envelope. This protein forms a homotrimer structure projecting out from its main body. This projected spike protein is an attractive target to generate antibodies and antiviral agents that will hampers it association with the host cell [8] . The SARS-CoV2 spike (S) protein comprises of two functional subunits, namely, S1 and S2 subunits. The S1 subunit comprises of N-terminal domain and the receptor binding domain (RBD) [9] . The S1 subunit of SARS-CoV2 first binds with the ACE receptor present on the host cell. The S2 subunit is composed of many regions that include fusion peptide (FP), heptad repeat 1, central helix, connector domain, heptad repeat 2, transmembrane domain, and cytoplasmic tail [10] (Fig 1A) . This subunit finally promotes the fusion the virus with the host cells. Coronaviruses get access into the host cell through their spikes on their surface. Ordinarily, S protein of coronaviruses is inactive prior to its binding with the host cell. Upon binding with the host cell receptor, coronaviruses achieve the ability to mediate their fusion with the host cell membrane by cleavage at S1/S2 cleavage site (CS) present in the S protein.

Depending upon the uniqueness of the respective amino acid sequence at S1/S2 CS in the S protein of coronaviruses, the cleavage is achieved by: (1) transmembrane serine-proteases (2) furin-like enzymes in the host cell (3) cathepsin proteases in the late endolysosome/endosome. Post S1/S2 site cleavage, a second cleavage site in the S2 domain of the spike protein gets exposed to serine-proteases or cathepsins, which subsequently drives the host-virus membrane fusion [11] . Cryo-electron microscopy performed on the SARS-CoV S protein bound with the ACE2 has revealed that initially S1 binds with the ACE2 receptor on the host cell receptor. This binding then drives the disassociation of S1 with the ACE2 receptor that consequently prompts the S2 to shift from a metastable prefusion to a more stable post-fusion state that drives the membrane fusion [8, 9, 12, 13] . In the prefusion conformational state, S1 and S2 subunits remain non-covalently bound. The S1 subunit comprising of the RBD houses receptor binding motif (RBM) (438−506) that interacts with the N-terminal peptidase domain of ACE receptor (Fig 1B) . SARS-CoV2 S protein exists in closed and the open conformational states. SARS-CoV2's RBD has higher binding affinity with the ACE2 receptor compared to SARS-CoV. SARS-CoV-2's F486 interacts with the Q24, L79, M82, and Y83 amino acids of the ACE2 receptor compared to SARS-CoV's L472 that interacts with the L79 and M82 amino acids of the ACE2 receptor ( Fig  1C) . The Q493 amino acid of the SARS-CoV2 RBD interacts with the ACE2's K31, E35, and H34 amino acids. In RBM, ACE's D30 forms a salt-bridge with the SARS-CoV-2 K417. Genetically engineered SARS-CoV2 RBD−ACE receptor complex comprising of RBM and the RBD core of the SARS-CoV S protein acts as a scaffold to promote crystallization. The side loop pointing away from the main binding region preserves a salt-bridge (N-O) between SARS-CoV-2 RBD's R426 and E329 of ACE receptor. The claw-like exposed structure of the ACE receptor binds with the concave SARS-CoV-2 RBM. The binding affinity of the ACE2 receptor with the SARS-CoV RBD is less compared to the SARS-CoV-2 and therefore, the infectivity and pathogenesis of SARS-CoV is less than the SARS-CoV2 however both recognizes the seventeen amino acid residues present in the human ACE2 receptor. In the closed state, the three RBM do not project out and all the three RBDs are facing down. In the open state, one of the RBD is in the upward conformation, which is necessary for the SARS-CoV-2 to fuse with the host cell [10] (Fig  1D, E) .

The secondary structure of the RBD consists of five β -strands organized in antiparallel fashion that is, in turn, connected by dominating loops (stabilized by disulphide linkage) and short α helices. Among the five β -strands, β 4 and β 7 consists of RBM, which in turn consists of α -helices, loops, and a short β 5 and β 6 strands. RBM is the portion of RBD that comprises of most of the binding sites for the human ACE2 receptor peptidase domain (19− 615) [10] . The SARS-CoV-2 S protein has nine cysteine residues and among them eight forms the four pair of disulphide linkage. The three disulphide linkage (C336-C361, C379-C432, and C391-C525) in the RBD increases the stability of β sheet present in it while as the C480-C488 linkage assists in promoting the connection between the loops present in the RBM. The crystallographic data has revealed the homotrimeric nature of the SARS-CoV2 S protein and among them one S protein's RBD is in up conformation or in open/ active state [14] . The binding of open conformation of the S protein with S1 subunit destabilizes the prefusion structure. Subsequently the S1 subunit detaches from the ACE2 receptor−S protein that promotes the S protein S2 subunit to refold into a stable post-fusion state. The SARS-CoV2 RBD straddles between a hinge-like structure that exposes or hides the RBD's active site to interact or disengage with the ACE receptor at the lung epithelial cells [10, 15] .

One of the remarkable features of the SARS-CoV-2 S protein is the presence of a four-amino acid sequence that starts with an amino acid acid proline (SPRRAR|S) that lies between the RBD (S1) and fusion (S2) domain [16] . This amino acid sequence is referred as a polybasic site and has been anticipated to be a part of the closely related origin of the SARS-CoV2 pandemic around the world [17] . Proteolytic cleavage of the S protein at its S1/S2 and S2 junction is extensively used to trigger the fusion apparatus of viral glycoproteins [11, 18] . SARS-CoV-2 S protein cleavage is not a simple process rather it involves a series of complex process comprising of more than one cleavage events at different sites coupled with the participation of wide range of host cell proteases [19] . Furin, a host cell protease, is ubiquitously expressed at low levels in Golgi apparatus of all cells. It plays an important role in virus-driven pathogenesis with its ability to cleave polybasic amino acid cleavage site (CS) found in some avian influenza virus and generally present in betacoronaviruses, thereby allowing its systemic dissemination. These polybasic sites are typically generated through polymerase slippage during replication of some avian influenza virus. Few viruses, such as influenza virus, are devoid of polybasic sites, and infection remains localized to those cells that possess trypsin-like proteases. Bat-origin MERS-like merbecovirises also harbors furin CS. Interestingly, it has been reported that the simple insertion of a polybasic site in H3 virus doesn't led to a high degree of pathogenicity and has been likely originated through a series of genetic changes during the course of a natural selection. However, as far as the origin and pathogenicity of SARS-CoV2 through recombination/mutation of a bat-origin virus has been understand as obscure [20] . The acquisition of furin cleavage motif between S1-S2 junction of SARS CoV2 S protein is astonishing and has led us to speculate that this virus has been genetically engineered. The furin cleavage sequence in SARS-CoV2 S protein doesn't resemble with the prototypical furin CS RxK/RR found in H3 virus. The wild-type SARS-CoV2 that first emerged had proline residue in furin CS. Subsequently, this residue was replaced by histidine in B.1.1.7 variant, and arginine in B.1.617 where tri-basic PRRAR/S sequence to RRRAR/S along with other adaptions. One can fairly infer that progressively these sites are becoming more polybasic with the continuous pandemic and increasing transmissibility that may consequently leads to the development of new variants [18] .

Progressive accumulation of mutation in the spike protein has added a layer complexity and has raised many questions such as how the S protein adapts to the environment of different species, tissue-types, and cell types. In the middle of the SARS-CoV2 S protein comprises of furin cleavage site for the subtilisin-identical host cell protease furin. Ordinarily host proteases, more particularly, TMPRSS cleaves the spike glycoprotein at the S2 site in order to activate the spike proteins for fusion with the host cell by achieving an irreversible conformational state. Furincleavage site makes SARS-CoV-2 S protein different from SARS-CoV S protein. This site comprises of four amino acid residues (P681, R682, R683, and A684) that is present at the boundary between the S1 and S2 subunit. Functionally, R682, R683, A684, and R685 create the nominal polybasic furin cleavage site (PRRAR). This sequence is not present in the previously known SARS-CoV and its related group 2b β -coronaviruses found in humans and this may be possibly one of the reasons for high virulence of SARS-CoV-2. Ubiquitously expressed furin proteases in humans are required for the activation of SARS-CoV-2 S protein. Genetically engineered furin-deleted SARS-CoV-2 spike protein has reduced processing and replication compared to the wild-type SARS-CoV-2 [11] .

This newly emerged SARS-CoV2 variant has recently been found circulating in South Africa and Botswana. Omicron virus is unique from the previously characterized SARS-CoV2 virus. It has been found that it has 37 amino acid substitutions in the S protein and 15 substitutions among them are located in RBD and 10 among them in RBM, a part of RBD that interacts with ACE2 receptor. Compared to the original wild type SARS-CoV2, the dominant Delta (B.1.617.2) variant has only 2 mutations (L452R and T478K) in its RBM and K417N and E484K mutations occasionally. Therefore, Omicron variant may significantly impact the binding affinity to ACE2. Consequently, Omicron mutant has aroused wide concern; many countries have taken measures on entry restrictions to prevent its rapid spread. However, the transmissibility and immune evasion risk of Omicron have not been properly evaluated [21] . The spike protein of WT SARS-CoV-2 has 1273 amino acids (UniProt ID: P0DTC2), and its RBD is composed of residues 319-541 and RBM is of residues 437-507. 2 The currently dominant Delta variant has only 4 mutations on its RBD (RBD Delta), much less than that on the Omicron RBD (RBD micron) (Fig. 1a ). It could be seen that the 15 mutations of RBD Omicron are not evenly distributed in RBD, but rather crowed in its RBM with 10 residues, viz., N440K, G446S, S477N, T478K, E484A, Q493K, G496S, Q498R, N501Y and Y505H (Fig. 1a) . By checking the effect of single mutation on ACE2 binding affinity reported by Bloom et al. 3 , it was found that 9 RBD Omicron mutations (S371L, S373P, S375F, K417N, G446S, E484A, G496S, Q498R, Y505H) should decrease the binding affinity to ACE2 while the other 6 mutations (G339D, N440K, S477N, T478K, Q493K, N501Y) should increase the binding affinity, resulting in a challenge of predicting its transmissibility and potential immune evasion risk [21, 22] . α

Alpha1 antitrypsin (α1AT ) is a 52kDa serine-protease inhibitor, which abundantly synthesized by hepatocytes and to some extent by other cells of the human body. Neutrophils move towards the site of inflammation, release neutrophil elastase (protease). However, in case of α 1AT deficiency, uncontrolled release of elastase leads to the manifestation of many diseases including parenchymal-degradation of lungs (emphysema). Therefore, α 1AT has an important role to neutralize the excessive production of elastase. X-ray crystallography, insilico analysis, and biochemical kinetics have provided us valuable insights to study its folding mechanism [23, 24] . During the initial folding process α 1ATis kinetically trapped into a metastable state along with a patch of 15 amino acid residues (345-360) located near to its the C-terminus that forms a flexible loop called as reactive-center loop (RCL), which is exposed to polar solvent. The conformational plasticity of α 1AT not only unveils its inhibitory activity but also provides a mechanistic understanding of pathogenic point-mutant 1AT is subsequently targeted for degradation [25] .

Reportedly, increased level of serum interleukin (IL)-6 cytokine has been observed in SARS-CoV2 infected patients who were observed to have moderate to severe symptoms. The IL-6/ α 1AT ratio indicates a hemeostatic switch between pro-and anti-inflammatory responses. This ratio was seen prominently high in SARS-CoV2 infected patients admitted in the intensive care unit compared to stable ones. α 1AT has been included as a potential clinical and biological biomarker marker under Clinical Trial number: NCT04348396 and NCT04366089 in Italy. Intriguingly, circulating levels of serum α 1AT in SARS-CoV2-infected patients were significantly low compared to healthy ones. Interestingly, truncated α 1AT mutant proteins were found to be significantly high in SARS-infected patients. The low bioavailablity of circulating α 1AT in SARS-infected patients were found to have lung failure and can lead to acute respiratory distress syndrome [26] . Potential role of α 1AT in SARS-CoV2 infected patients α 1AT alleviates the acute lung damage by decreasing the IL-1β levels. It stands guard against the thrombotic complications such as macrothrombi and small vessel thrombosis observed in SARS-CoV2 infected patients. Thrombotic complications in SARS-CoV2-infected patients can lead to disease progression, organ failure, and poor outcome. It inhibits the cytokine storm and its augmentation therapy acts as an immune modulator to dampen the pro-inflammatory IL-6 cytokine levels and triggers balanced antiviral immune response that subsequently leads to the efficacious pathogen clearance without tissue damage. It sequesters IL-8 that consequently dampens the neutrophil influx and acute-lung damage and alleviates TGF-β-driven abrupt and longstanding damaging effects of SARS-CoV2. Accumulating investigations suggests conceivable association between α 1AT and SARS-CoV2. FDA has approved α 1AT as one of the drug that can be used with extraordinary clinical safety [27] .

TMPRSS2 is a 492 amino acid residue long single-pass type II membrane protein. It comprises of N-terminus domain located in the cytosol, TM domain, and a cytosolic domain. The cytosolic domain is further divided into three sub regions, namely LDL receptor class A from 113−148 amino acid that serves as a binding site for calcium, scavenger receptor cysteine-rich domain (from 149−242 amino acid residues), and a serine-protease (SP) domain extending from 255−492 amino acid residues of the S1 family.

The SP domain contains the H296, D354, and S441 that acts a catalytic triad. It is exclusively expressed at the cell surface and serves to regulate the cell-cell and cell matrix interaction. It is expressed in normal as well as in diseased condition. It is highly expressed in small intestine and to small extent in salivary gland, stomach, colon, and prostate gland. This protein is upregulated by androgenic hormones secreted by prostate cancer cells. It acts a receptor for specific ligands that helps it to coordinate with the extracellular environment of the cell. In lungs, TMPRSS2 has proposed to regulate the sodium current of the epithelial cells cleaving the sodium channel. It facilitates the SARS-CoV and SARS-CoV-2 infection in the host cells through two independent mechanisms, ACE2 receptor proteolytic cleavage that promotes viral infection, and cleavage of the SARS-CoV-2 S protein which activates the SARS-CoV-2 to get an access into the host cell. α 1AT antiprotease inhibits the SARS-CoV2 infection by deactivating the TMPRSS2 protease activity. SARS-CoV-2 primarily spreads through inhalation of droplets and aerosols coming from the infected person and reinfection of respiratory tract cells with the same virus. In overwhelming majority of the SARS-CoV2 infected cases, infection remains confined to the upper airways causing no or mild symptoms. Chronic disease driven by SARS-CoV-2 is caused by the viral dissemination down to the lungs, consequently leading to the acute respiratory distress syndrome, cytokine burst, multi-organ failure, systemic septic shock, and death. The airway epithelium acts as a first-line of defence against the invading respiratory microorganisms through the mucociliary clearance action and its mucosal-associated lymphoid tissue. The epithelial lining fluid comprises of innate immunity effector molecules like short-peptides as well as proteins that act as antibacterial and antiviral agents, such as lactoferrin or defensins, lysozyme, and α 1AT protein.

Recently, it has been demonstrated that α 1AT, a circulating serine protease inhibitor, stops the entry of SARS-CoV-2 in human airway epithelial by binding with TMPRSS2 antiprotease and deactivating it. Active TMPRSS2 activates the SARS-CoV-2 spike protein for its fusion with the human airway epithelial cells. In vitro experiments have revealed that α 1AT protein inhibits the SARS-CoV-2 infection with IC 50 of 10-20µM. Therefore, one would suggest that α 1AT has anti-SARS-CoV-2 activity. It wouldn't be surprising to suggest that α 1AT deficient individuals are highly susceptible to SARS-CoV2 infection, more specifically, in early-onset COPD patients. Intravenous α 1AT augmentation therapy has been used from decades in individuals suffering from α 1AT deficiency driven early onset of parenchymal lung disease (emphysema). Therefore, α 1AT augmentation therapy can prove instrumental by acting as an antinflamatory protein as well as anti-SARS-CoV2 agent. α 1AT can be administered in body at significantly higher doses than those in routine α 1AT deficiency. Interestingly, in some α 1AT deficient patients, α 1AT administered at doses of 120−250 mg/kg without causing any side effects, leads to a five-fold increase of α 1AT concentration in lung epithelial lining fluid has been observed. However, it is still unclear whether α 1AT infusion in human body will achieve a threshold concentration that is sufficient to block the entry of SARS-CoV-2 in lungs without leading to undesirable severe side effects remains yet to uncovered [26] .

Protein Homology/analogy Recognition Engine V 2.0 (Phyre 2 ) was used for modeling the SARS-CoV2 S protein with a template input available structure of S protein with PDB ID: 6vyb. The modeled S protein with dimension ( ): X=74.777, Y=124.039, Z=159.510 was built on template c7dk3B with 100% confidence and 83% coverage. 1062 residues (83% of the sequence) was modeled with 100% confidence by the single highest scoring template. 1141 residues (90%) was modeled at >90% confidence using multi-templates [28] .

Spike protein Arg/Lys cleavage prediction at S1/S2 furin cleavage site Propeptide cleavage sites predicted (ProP): Arg(R)/Lys (K): 3. If the score is >0.5, the residue is predicted to be followed by propeptide cleavage site.The higher the score the more confident the prediction. Prediction scores for the S1/S2 furin cleavage site in S protein were analyzed by using the ProP 1.0 server (www.cbs.dtu.dk/services/ProP/) [29] The prepared proteins were docked through https://cluspro.bu.edu. [30] [31] [32] 

We tried to observe the electrostatic potential of SARS-CoV2 RBD mutants complexed with the ACE2 receptor at their binding interface. We observed a dramatic change in the electrostatic potential of SARS-CoV2 RBD (K417N) and SARS-CoV2 RBD triple mutant (K417N, E484K, N501Y) complexed with the ACE2 receptor at their binding interface compared to the wild-type SARS-CoV2 RBD (Fig 2A-H) . Our results revealed that single and triple-mutants of SARS-CoV2 RBD have different electrostatic potential at their binding interface. 14 amino acid missing residues form antiparallel β -sheet harboring furin cleavage site. Phylogenetic investigation of the newly evolved SARS-CoV-2 is unique in a manner that it has an insertion of RRAR sequence/furin cleavage site at the S1/S2 site of SARS-CoV2 S protein compared to previously identified SARS-CoV and other SARS-related coronaviruses. The addition of this sequence in SARS-CoV2 has been speculated to provide a gain-of-function, thereby increasing its efficacy of transmission in humans by easily gaining an access to airway epithelial cells compared to other lineage B β -coronaviruses that eventually leads to a host of clinical condition such as lung damage, bilobular-pneumonia, macrothrombi formation etc. N-terminal domain and RBD harboring RBM are present in the S1 subunit of SARS-CoV-2 S protein. The S2 subunit of SARS-CoV-2 consists of fusion peptide, heptad repeat 1, central helix, connector domain, heptad repeat 2, transmembrane domain, and a tail flanking towards the cytosol (Fig.3A) . The furin cleavage site resides between these two domains. In overwhelming majority of the coronavirus, host proteases like TMPRSS2 cleave the FCS through an irreversible conformational change that assists in the viral fusion with the host cell. Majority of investigators have reported the presence of a loop between S1/S2 junction of SARS-CoV2 S protein. In this connection, we modeled full SARS-CoV2 spike protein through Protein Homology/ analogy Y Recognition Engine V 2.0) by taking the available SARS-CoV2 spike protein with PDB: 6VYB as a template. SARS-CoV2 S protein was modeled because of the reason that the available structure lacks a nine amino acid patch (677QTNSPRRAR685) that essentially provides a furin cleavage site. We found that this sequence forms two anti-parallel β -sheets comprising of FCS that protrudes out of the main body of SARS-CoV2 S protein (Fig 3C) . The electrostatic potential of the available and modeled SARS-CoV2 S protein is shown in Fig3. B, D. The secondary structure of our modeled SARS-CoV2 S protein has 37% in β strand, 26% in α helices, 16% in a disordered state, and 7% existing as transmembrane (Fig 3E.) . We anticipate that this β -sheet is used as a scaffold by proteases to act on furin-CS in SARS-CoV2 S protein.

We found that SARS-CoV2 S protein comprising of furin cleavage site (682RRAR685 ) interacts with the D417, N418, Y416, and N418 amino acid residues of the TMPRSS2 protease (Fig 4D, E) . The electrostatic surface potential of the SARS-CoV2 S protein TMPRSS2 protease is shown in Fig 4F. 

We found that SARS-CoV2 S protein comprising of furin cleavage site (681PRR-R685 ) interacts with the S368, D258, E257, and E236 amino acid residues of the furin protease (Fig 4G, H) . The electrostatic surface potential of the SARS-CoV2 S protein furin protease is shown in Fig 4I. Arg/Lys cleavage prediction at S1/S2 furin cleavage site in SARS-CoV2 S protein We analysed the Arginine (Arg)/Lysine (Lys) cleavage prediction at S1/S2 furin cleavage site in SARS-CoV2 S protein. The propeptide cleavage sites predicted Proline: Arg/Lys ratio 3, pointing the three prominent Arg/Lys cleavage sites in SARS-CoV2 S protein at around between 400-600, 600-800, and 800-820 amino acid residues (Fig 4M) . 1AT acts as an antiprotease effector protein to dampen the immune response driven by the activation of NE during microbial invasion that leads to inflammation. TMPRSS2 binds with the SARS-CoV2 S proteins S1 subunit bound with the ACE receptor present on airway epithelial cells. The bound TMPRSS2 cleaves the S protein at S1 site. We found that α 1AT by virtue of its RCL interacts with the TMPRSS2 and deactivates its protease activity (Fig 4A, B, C) .

α 1AT antiprotease interacts with furin protease. We observed that α 1AT uses its RCL to interact and subsequently deactivate the furin protease (Fig 4J,  J) . The electrostatic potential of the complex is shown in Fig 4L. 

Majority of investigators have reported the presence of a loop harboring the furin cleavage site of SARS-CoV2 S protein. We are for the first time reporting this patch comprises of 14 amino acid residues (677QTNSPRRARSVASQ689) that form an antiparallel β -sheet comprising of already known PRRAR furin polybasic CS. We anticipate that this β -sheet is used as a scaffold by proteases to act on furin-CS in SARS-CoV2 S protein. Additionally, we studied the interaction of modeled SARS-CoV2 S protein with transmembrane protease, serine 2 (TMPRSS2), and furin proteases that accentuated that these proteases use furin CS sequence (PRRAR) located in β -sheet of our modeled SARS-CoV2 S protein to cleave the SARS-CoV2 S protein at S1/S2 junction.

The idea and proposal of the study was designed by Arif Bashir Arg/Lys cleavage prediction at S1/S2 furin cleavage site. Propeptide cleavage sites predicted Proline: Arginine/Lysine: 3. If the score is >0.5, the residue is predicted to be followed by propeptide cleavage site. The higher the score the more confident the prediction. Prediction scores for the S1/S2 furin cleavage site in S protein were analyzed by using the ProP 1.0 server (www.cbs.dtu.dk/services/ProP/).

A pneumonia outbreak associated with a new coronavirus of probable bat origin

A new coronavirus associated with human respiratory disease in China

A Novel Coronavirus from Patients with Pneumonia in China

Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding

SARS-CoV-2 variants, spike mutations and immune escape

SARS-CoV-2 evolution in animals suggests mechanisms for rapid variant selection

SARS-CoV-2 Quasispecies Provides an Advantage Mutation Pool for the Epidemic Variants. Microbiol Spectr

Cryo-electron microscopy structures of the SARS-CoV spike glycoprotein reveal a prerequisite conformational state for receptor binding

Stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor

The furin cleavage site in the SARS-CoV-2 spike protein is required for transmission in ferrets

Cryo-EM structures of MERS-CoV and SARS-CoV spike glycoproteins reveal the dynamic receptor binding domains

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Receptor Recognition by the Novel Coronavirus from Wuhan: an Analysis Based on Decade-Long Structural Studies of SARS Coronavirus

The proximal origin of SARS-CoV-2

Characterization of a highly conserved domain within the severe acute respiratory syndrome coronavirus spike protein S2 domain with characteristics of a viral fusion peptide

SARS-CoV-2 spike and its adaptable furin cleavage site

Coronavirus Spike Protein and Tropism Changes

Furin Cleavage Site Is Key to SARS-CoV-2 Pathogenesis. bioRxiv

Insights on the mutational landscape of the SARS-CoV-2 Omicron variant. bioRxiv

SARS-CoV-2 Omicron RBD shows weaker binding affinity than the currently dominant Delta variant to human ACE2

Dynamic local unfolding in the serpin alpha-1 antitrypsin provides a mechanism for loop insertion and polymerization

Folding mechanism of the metastable serpin alpha1-antitrypsin

Aggregation of M3 (E376D) variant of alpha1-antitrypsin

Alpha-1 antitrypsin inhibits TMPRSS2 protease activity and SARS-CoV-2 infection

Alpha 1 Antitrypsin is an Inhibitor of the SARS-CoV-2-Priming Protease TMPRSS2. Pathog Immun

The Phyre2 web portal for protein modeling, prediction and analysis

Prediction of proprotein convertase cleavage sites

Performance and Its Limits in Rigid Body Protein-Protein Docking

New additions to the ClusPro server motivated by CAPRI

The ClusPro web server for protein-protein docking

Ligand docking and binding site analysis with PyMOL and Autodock/Vina

We declare no conflict of interest.