Heparin Inhibits Cellular Invasion by SARS-CoV-2: Structural Dependence of the Interaction of the Spike S1 Receptor-Binding Domain with Heparin

AbstractThe dependence of development and homeostasis in animals on the interaction of hundreds of extracellular regulatory proteins with the peri- and extracellular glycosaminoglycan heparan sulfate (HS) is exploited by many microbial pathogens as a means of adherence and invasion. Heparin, a widely used anticoagulant drug, is structurally similar to HS and is a common experimental proxy. Exogenous heparin prevents infection by a range of viruses, including S-associated coronavirus isolate HSR1. Here, we show that heparin inhibits severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) invasion of Vero cells by up to 80% at doses achievable through prophylaxis and, particularly relevant, within the range deliverable by nebulisation. Surface plasmon resonance and circular dichroism spectroscopy demonstrate that heparin and enoxaparin, a low-molecular-weight heparin which is a clinical anticoagulant, bind and induce a conformational change in the spike (S1) protein receptor-binding domain (S1 RBD) of SARS-CoV-2. A library of heparin derivatives and size-defined fragments were used to probe the structural basis of this interaction. Binding to the RBD is more strongly dependent on the presence of 2-O or 6-O sulfate groups than on N-sulfation and a hexasaccharide is the minimum size required for secondary structural changes to be induced in the RBD. It is likely that inhibition of viral infection arises from an overlap between the binding sites of heparin/HS on S1 RBD and that of the angiotensin-converting enzyme 2. The results suggest a route for the rapid development of a first-line therapeutic by repurposing heparin and its derivatives as antiviral agents against SARS-CoV-2 and other members of the Coronaviridae.

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Heparin inhibits cellular invasion by SARS-CoV-2: structural dependence of the interaction of the surface protein (spike) S1 receptor binding domain with heparin

10.1101/2020.04.28.066761 ◽

2020 ◽

Cited By ~ 26

Author(s):

Courtney J. Mycroft-West ◽

Dunhao Su ◽

Isabel Pagani ◽

Timothy R. Rudd ◽

Stefano Elli ◽

...

Keyword(s):

Conformational Change ◽

Receptor Binding ◽

Conformational Changes ◽

Rapid Development ◽

Heparan Sulphate ◽

Binding Domain ◽

Extracellular Proteins ◽

Microbial Pathogens ◽

Receptor Binding Domain ◽

Heparin Binding

AbstractThe dependence of the host on the interaction of hundreds of extracellular proteins with the cell surface glycosaminoglycan heparan sulphate (HS) for the regulation of homeostasis is exploited by many microbial pathogens as a means of adherence and invasion. The closely related polysaccharide heparin, the widely used anticoagulant drug, which is structurally similar to HS and is a common experimental proxy, can be expected to mimic the properties of HS. Heparin prevents infection by a range of viruses if added exogenously, including S-associated coronavirus strain HSR1. Heparin prevents infection by a range of viruses if added exogenously, including S-associated coronavirus strain HSR1. Here, we show that the addition of heparin to Vero cells between 6.25 - 200 μg.ml−1, which spans the concentration of heparin in therapeutic use, and inhibits invasion by SARS-CoV-2 at between 44 and 80%. We also demonstrate that heparin binds to the Spike (S1) protein receptor binding domain and induces a conformational change, illustrated by surface plasmon resonance and circular dichroism spectroscopy studies. The structural features of heparin on which this interaction depends were investigated using a library of heparin derivatives and size-defined fragments. Binding is more strongly dependent on the presence of 2-O or 6-O sulphation, and the consequent conformational consequences in the heparin structure, than on N-sulphation. A hexasaccharide is required for conformational changes to be induced in the secondary structure that are comparable to those that arise from heparin binding. Enoxaparin, a low molecular weight clinical anticoagulant, also binds the S1 RBD protein and induces conformational change. These findings have implications for the rapid development of a first-line therapeutic by repurposing heparin as well as for next-generation, tailor-made, GAG-based antiviral agents against SARS-CoV-2 and other members of the Coronaviridae.

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Glycosaminoglycans induce conformational change in the SARS-CoV-2 Spike S1 Receptor Binding Domain

10.1101/2020.04.29.068767 ◽

2020 ◽

Cited By ~ 4

Author(s):

Courtney J. Mycroft-West ◽

Dunhao Su ◽

Yong Li ◽

Scott E. Guimond ◽

Timothy R. Rudd ◽

...

Keyword(s):

Conformational Change ◽

Receptor Binding ◽

Conformational Changes ◽

Heparan Sulphate ◽

Antiviral Agents ◽

Structural Features ◽

Binding Domain ◽

Microbial Pathogens ◽

Future Research ◽

Receptor Binding Domain

AbstractThe glycosaminoglycan (GAG) class of polysaccharides are utilised by a plethora of microbial pathogens as receptors for adherence and invasion. The GAG heparin prevents infection by a range of viruses when added exogenously, including the S-associated coronavirus strain HSR1 and more recently we have demonstrated that heparin can block cellular invasion by SARS-CoV-2. Heparin has found widespread clinical use as anticoagulant drug and this molecule is routinely used as a proxy for the GAG, heparan sulphate (HS), a structural analogue located on the cell surface, which is a known receptor for viral invasion. Previous work has demonstrated that unfractionated heparin and low molecular weight heparins binds to the Spike (S1) protein receptor binding domain, inducing distinct conformational change and we have further explored the structural features of heparin with regard to these interactions. In this article, previous research is expanded to now include a broader range of GAG family members, including heparan sulphate. This research demonstrates that GAGs, other than those of heparin (or its derivatives), can also interact with the SARS-CoV-2 Spike S1 receptor binding domain and induce distinct conformational changes within this region. These findings pave the way for future research into next-generation, tailor-made, GAG-based antiviral agents, against SARS-CoV-2 and other members of the Coronaviridae.

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Gonococcal pili. Primary structure and receptor binding domain.

Journal of Experimental Medicine ◽

10.1084/jem.159.5.1351 ◽

1984 ◽

Vol 159 (5) ◽

pp. 1351-1370 ◽

Cited By ~ 79

Author(s):

G K Schoolnik ◽

R Fernandez ◽

J Y Tai ◽

J Rothbard ◽

E C Gotschlich

Keyword(s):

Amino Acids ◽

Receptor Binding ◽

Binding Domain ◽

Structural Basis ◽

Receptor Binding Domain ◽

Variable Regions ◽

Disulfide Linkage ◽

Polymeric Structure ◽

Antigenic Diversity ◽

Single Polypeptide Chain

The complete amino acid sequence of pilin from gonococcal strain MS11 and the sequence of constant and variable regions from strain R10 pilin have been determined in order to elucidate the structural basis for adherence function, antigenic diversity, and polymeric structure. The MS11 pilin sequence consists of 159 amino acids in a single polypeptide chain with two cysteines in disulfide linkage and serine-bonded phosphate residues. TC-2 (31-111), a soluble monomeric pilus peptide prepared by arginine-specific digestion, bound human endocervical, but not buccal or HeLa cells and therefore is postulated to encompass the receptor binding domain. Variable regions of CNBr-3 appear to confer antigenic diversity and comprise segments in which changes in the position of charged residues occur in hydrophilic, beta-turns. Residues 2-21 and 202-221 of gonococcal pilins and lower eucaryotic actins, respectively, exhibit 50% homology. When these residues are arranged at intervals of 100 degrees of arc on "helical wheels," the identical amino acids comprise a hydrophobic face on one side of the helix. This observation, the hydrophobic character of this region and the tendency for TC-1 (residues 1-30) to aggregate in water, suggest that this stretch interacts with other subunits to stabilize polymeric structure.

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Towards a Recombinant Vaccine against Diphtheria Toxin

Infection and Immunity ◽

10.1128/iai.66.2.418-423.1998 ◽

1998 ◽

Vol 66 (2) ◽

pp. 418-423 ◽

Cited By ~ 15

Author(s):

Karin Lobeck ◽

Pascal Drevet ◽

Michel Léonetti ◽

Cécile Fromen-Romano ◽

Frédéric Ducancel ◽

...

Keyword(s):

Receptor Binding ◽

Diphtheria Toxin ◽

Protein A ◽

Vero Cells ◽

Binding Domain ◽

Receptor Binding Domain ◽

Surface Receptors ◽

Linear Peptide ◽

Recombinant Fragments

ABSTRACT Two recombinant fragments of diphtheria toxin (DT) were fused to an engineered tandem repeat of the immunoglobulin (Ig) binding domain of protein A, called ZZ. These fragments are (i) the receptor binding domain (DTR), which comprises amino acids 382 to 535 of DT, and (ii) a linear peptide (DT168–220) which comprises residues 168 to 220 of the loop between fragment A and fragment B of DT. The fusion proteins were produced in Escherichia coli and purified by affinity chromatography. In vitro experiments showed that the DTR domain is responsible for the capacity of ZZ-DTR to bind to Vero cells and is capable of inhibiting the cytotoxicity of DT for these cells. These findings suggest that DTR binds to the cell surface receptors of DT and hence adopts a conformation that is similar to that of the receptor binding domain of DT. We compared the capacities of ZZ-DTR, ZZ-DT168–220, and a chemically detoxified form of DT currently used for vaccination to elicit antibodies in rabbits. The toxoid was more immunogenic than ZZ-DT168–220, which in turn was more immunogenic than ZZ-DTR. However, ZZ-DT168–220 antiserum was poorly efficient at neutralizing DT cytotoxicity on Vero cells, whereas ZZ-DTR antiserum was only 15-fold less potent than anti-DT antisera.

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A single de novo substitution in SARS-CoV-2 spike informs enhanced adherence to human ACE2.

10.1101/2021.07.16.452441 ◽

2021 ◽

Author(s):

Elena Erausquin ◽

Jacinto Lopez-Sagaseta

Keyword(s):

Amino Acid ◽

Cell Membrane ◽

Receptor Binding ◽

De Novo ◽

Binding Domain ◽

Host Cells ◽

Structural Basis ◽

Receptor Binding Domain ◽

To Come ◽

Single Substitution

SARS-CoV-2 initiates colonization of host cells by binding to cell membrane ACE2 receptor. This binding is mediated by the viral spike receptor binding domain (RBD). The COVID-19 pandemic has brought devastating consequences at a clinical, social and economical levels. Therefore, anticipation of potential novel SARS-causing species or SARS-CoV-2 variants with enhanced binding to ACE2 is key in the prevention of future threats to come. We have characterized a de novo single substitution, Q498Y, in SARS-CoV-2 RBD that confers stronger adherence to ACE2. While the SARS-CoV-2 beta variant, which includes three simultaneous amino acid replacements, induces a 4-fold stronger affinity, a single Q498Y substitution results in 2.5-fold tighter binding, compared to the Wuhan-Hu-1 SARS-CoV-2 2019 strain. Additionally, we crystallized RBDQ498Y complexed with ACE2 and provide here the structural basis for this enhanced affinity. These studies inform a rationale for prevention of potential SARS-causing viruses to come.

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SARS-CoV-2 Variant of Concern Substitutions Alter Spike Glycoprotein Receptor Binding Domain Structure and Stability

10.1101/2021.05.11.443443 ◽

2021 ◽

Author(s):

Daniel L Moss ◽

Jay Rappaport

Keyword(s):

Receptor Binding ◽

Rapid Development ◽

Structure And Function ◽

Binding Domain ◽

Amino Acid Substitutions ◽

Receptor Binding Domain ◽

Spike Glycoprotein ◽

Structure And Stability ◽

The World ◽

And Function

The emergence of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and the subsequent COVID-19 pandemic has significantly impacted the world not just with disease and death but also economic turmoil. The rapid development and deployment of extremely effective vaccines against SARS-CoV-2 has made the end of the pandemic a reality within reach. However, as the virus spreads it has acquired mutations; and thus, variants of concern have emerged that are more infectious and reduce the efficacy of existing vaccines. While promising efforts are underway to combat these variants, the evolutionary pressures leading to these variants are poorly understood. To that end, here we have studied the effects of three amino-acid substitutions on the structure and function of the SARS-CoV-2 spike glycoprotein receptor-binding domain found in several variants of concern such as B.1.1.7, B.1.351 and P.1 that are now circulating. We found that these substitutions alter the RBD structure and stability, as well as its ability to bind to ACE2, which may have opposing and compensatory effects. These findings provide new insights into how these Variants of Concern (VOC) may have been selected to optimize infectivity while maintaining the structure and stability of the receptor binding domain.

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Development of a COVID-19 vaccine based on the receptor binding domain displayed on virus-like particles

10.1101/2020.05.06.079830 ◽

2020 ◽

Cited By ~ 6

Author(s):

Lisha Zha ◽

Hongxin Zhao ◽

Mona O. Mohsen ◽

Liang Hong ◽

Yuhang Zhou ◽

...

Keyword(s):

Receptor Binding ◽

Vaccine Candidate ◽

Rapid Development ◽

Binding Domain ◽

Spike Protein ◽

Receptor Binding Domain ◽

Viral Receptor ◽

Virus Like Particles ◽

The City

AbstractThe recently ermerging disease COVID-19 is caused by the new SARS-CoV-2 virus first detected in the city of Wuhan, China. From there it has been rapidly spreading inside and outside China. With initial death rates around 4%, COVID-19 patients at longer distances from Wuhan showed reduced mortality as was previously observed for the SARS coronavirus. However, the new coronavirus spreads more strongly, as it sheds long before onset of symptoms or may be transmitted by people without symptoms. Rapid development of a protective vaccine against COVID-19 is therefore of paramount importance. Here we demonstrate that recombinantly expressed receptor binding domain (RBD) of the spike protein homologous to SARS binds to ACE2, the viral receptor. Higly repetitive display of RBD on immunologically optimized virus-like particles derived from cucumber mosaic virus resulted in a vaccine candidate (RBD-CuMVTT) that induced high levels of specific antibodies in mice which were able to block binding of spike protein to ACE2 and potently neutralized the SARS-CoV-2 virus in vitro.

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The 2019 coronavirus (SARS-CoV-2) surface protein (Spike) S1 Receptor Binding Domain undergoes conformational change upon heparin binding

10.1101/2020.02.29.971093 ◽

2020 ◽

Cited By ~ 50

Author(s):

Courtney Mycroft-West ◽

Dunhao Su ◽

Stefano Elli ◽

Yong Li ◽

Scott Guimond ◽

...

Keyword(s):

Receptor Binding ◽

Rapid Development ◽

Heparan Sulphate ◽

Surface Protein ◽

Binding Domain ◽

Extracellular Proteins ◽

Receptor Binding Domain ◽

Heparin Binding ◽

Protein Receptor ◽

Surface Receptor

AbstractMany pathogens take advantage of the dependence of the host on the interaction of hundreds of extracellular proteins with the glycosaminoglycans heparan sulphate to regulate homeostasis and use heparan sulphate as a means to adhere and gain access to cells. Moreover, mucosal epithelia such as that of the respiratory tract are protected by a layer of mucin polysaccharides, which are usually sulphated. Consequently, the polydisperse, natural products of heparan sulphate and the allied polysaccharide, heparin have been found to be involved and prevent infection by a range of viruses including S-associated coronavirus strain HSR1. Here we use surface plasmon resonance and circular dichroism to measure the interaction between the SARS-CoV-2 Spike S1 protein receptor binding domain (SARS-CoV-2 S1 RBD) and heparin. The data demonstrate an interaction between the recombinant surface receptor binding domain and the polysaccharide. This has implications for the rapid development of a first-line therapeutic by repurposing heparin and for next-generation, tailor-made, GAG-based antivirals.

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Cryo-EM Structures of the N501Y SARS-CoV-2 Spike Protein in Complex with ACE2 and Two Potent Neutralizing Antibodies

10.1101/2021.01.11.426269 ◽

2021 ◽

Cited By ~ 1

Author(s):

Xing Zhu ◽

Dhiraj Mannar ◽

Shanti S. Srivastava ◽

Alison M. Berezuk ◽

Jean-Philippe Demers ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Structural Changes ◽

Neutralizing Antibody ◽

Antibody Fragments ◽

Binding Domain ◽

Spike Protein ◽

Receptor Binding Domain ◽

Binding Interface ◽

Short Summary

AbstractThe recently reported “UK variant” of SARS-CoV-2 is thought to be more infectious than previously circulating strains as a result of several changes, including the N501Y mutation. We present a 2.9-Å resolution cryo-EM structure of the complex between the ACE2 receptor and N501Y spike protein ectodomains that shows Y501 inserted into a cavity at the binding interface near Y41 of ACE2. The additional interactions result in increased affinity of ACE2 for the N501Y mutant, accounting for its increased infectivity. However, this mutation does not result in large structural changes, enabling important neutralization epitopes to be retained in the spike receptor binding domain. We confirmed this through biophysical assays and by determining cryo-EM structures of spike protein ectodomains bound to two representative potent neutralizing antibody fragments.Short summaryThe N501Y mutation found in the coronavirus UK variant increases infectivity but some neutralizing antibodies can still bind.

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Structures of synthetic nanobody–SARS-CoV-2–RBD complexes reveal distinct sites of interaction and recognition of variants

10.21203/rs.3.rs-625642/v1 ◽

2021 ◽

Author(s):

David Margulies ◽

Javeed Ahmad ◽

Jiansheng Jiang ◽

Lisa Boyd ◽

Allison Zeher ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Crystal Structures ◽

Receptor Binding ◽

Ternary Complexes ◽

Binding Domain ◽

Structural Basis ◽

Receptor Binding Domain ◽

S Protein ◽

X Ray ◽

Binary And Ternary Complexes

Abstract The worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and emergence of new variants demands understanding the structural basis of the interaction of antibodies with the SARS-CoV-2 receptor-binding domain (RBD). Here we report five X-ray crystal structures of sybodies (synthetic nanobodies) including binary and ternary complexes of Sb16–RBD, Sb45–RBD, Sb14–RBD–Sb68, and Sb45–RBD–Sb68; and Sb16 unliganded. These reveal that Sb14, Sb16, and Sb45 bind the RBD at the ACE2 interface and that the Sb16 interaction is accompanied by a large CDR2 shift. In contrast, Sb68 interacts at the periphery of the interface. We also determined cryo-EM structures of Sb45 bound to spike (S). Superposition of the X-ray structures of sybodies onto the trimeric S protein cryo-EM map indicates some may bind both "up" and "down" configurations, but others may not. Sensitivity of sybody binding to several recently identified RBD mutants is consistent with these structures.

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