scholarly journals High-resolution asymmetric structure of a Fab–virus complex reveals overlap with the receptor binding site

2021 ◽  
Vol 118 (23) ◽  
pp. e2025452118
Author(s):  
Daniel J. Goetschius ◽  
Samantha R. Hartmann ◽  
Lindsey J. Organtini ◽  
Heather Callaway ◽  
Kai Huang ◽  
...  
Keyword(s):  
High Resolution ◽  
Binding Site ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Antigenic Site ◽  
Conformational Epitope ◽  
Asymmetric Structure ◽  
Antigenic Epitope ◽  
Receptor Binding Site ◽  
Virus Complex

Canine parvovirus is an important pathogen causing severe diseases in dogs, including acute hemorrhagic enteritis, myocarditis, and cerebellar disease. Overlap on the surface of parvovirus capsids between the antigenic epitope and the receptor binding site has contributed to cross-species transmission, giving rise to closely related variants. It has been shown that Mab 14 strongly binds and neutralizes canine but not feline parvovirus, suggesting this antigenic site also controls species-specific receptor binding. To visualize the conformational epitope at high resolution, we solved the cryogenic electron microscopy (cryo-EM) structure of the Fab–virus complex. We also created custom software, Icosahedral Subparticle Extraction and Correlated Classification, to solve a Fab–virus complex with only a few Fab bound per capsid and visualize local structures of the Fab-bound and -unbound antigenic sites extracted from the same complex map. Our results identified the antigenic epitope that had significant overlap with the receptor binding site, and the structures revealed that binding of Fab induced conformational changes to the virus. We were also able to assign the order and position of attached Fabs to allow assessment of complementarity between the Fabs bound to different positions. This approach therefore provides a method for using cryo-EM to investigate complementarity of antibody binding.

scholarly journals Mechanisms of antibody binding revealed by asymmetric Fab-virus complexes

2020 ◽  
Author(s):  
Daniel J. Goetschius ◽  
Samantha R. Hartmann ◽  
Lindsey J. Organtini ◽  
Heather Callaway ◽  
Kai Huang ◽  
...  
Keyword(s):  
High Resolution ◽  
Binding Site ◽  
Receptor Binding ◽  
Antibody Binding ◽  
Antigenic Epitope ◽  
Receptor Binding Site ◽  
Local Structures ◽  
Antigenic Sites ◽  
High Resolution Map ◽  
Feline Parvovirus

AbstractOverlap on the surface of parvovirus capsids between the antigenic epitope and the receptor binding site contributes to species jumping. Mab 14 strongly binds and neutralizes canine, but not feline parvovirus. The high resolution map of the canine parvovirus capsid complexed with Fab 14 was used to solve local structures of the Fab-bound and -unbound antigenic sites extracted from the same complex. The subsequent analysis includes a new method for using cryo EM to investigate complementarity of antibody binding.

scholarly journals Broad Neutralization of Viral Pathogens

2014 ◽  
Vol 70 (a1) ◽  
pp. C245-C245
Author(s):  
Ian Wilson
Keyword(s):  
Hepatitis C ◽  
Binding Site ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Functional Characterization ◽  
Antigenic Site ◽  
Soluble Form ◽  
Receptor Binding Site ◽  
Fusion Domain ◽  
Hiv 1

Influenza, Hepatitis C, and HIV-1 continue to constitute significant threats to global health. We have structurally and functionally characterized several potent, broadly neutralizing antibodies (bnAbs) against HIV-1, influenza and hepatitis C viruses. The surface antigens of these viruses are the main target of neutralizing antibodies. However, most antibodies are strain-specific and protect only against highly related strains within the same subtype. Recently, a number of antibodies have been identified that are much broader and neutralize across multiple subtypes and types of these viruses through binding to functionally conserved sites, such as the receptor binding site or the fusion domain. For example, co-crystal structures of bnAbs with influenza hemagglutinin (HA) identified highly conserved sites in the fusion domain (stem) and in the receptor binding site (head) as target for broad neutralization[1]. HCV is also genetically diverse, but some antibodies have potent neutralizing activity across most genotypes of the virus. One family of these antibodies targets a conserved antigenic site on the HCV E2 envelope glycoprotein that overlaps with the CD81 receptor-binding site[2]. For HIV-1, structural and functional characterization of different families of bnAbs have led to identification of novel epitopes on HIV-1 Env, many of which involve glycans. These glycan-dependent Abs have unique features that enable them to penetrate the glycan shield and bind complex epitopes that consist of sugars and underlying protein segments on gp120 on HIV-1 Env. Recent x-ray[3] and EM structures of a soluble form of HIV-1 Env have revealed that the epitopes are more extensive and complex than previously appreciated. This structural information is now being used to aid in structure-assisted vaccine design for HIV-1, HCV and for a more universal flu vaccine. IAW is supported by NIH grants AI100663, AI082362, AI84817, AI099275 and GM094586 and the Crucell Vaccine Institute.

scholarly journals High-Resolution Definition of Vaccine-Elicited B Cell Responses Against the HIV Primary Receptor Binding Site

2012 ◽  
Vol 4 (142) ◽  
pp. 142ra96-142ra96 ◽  
Author(s):  
C. Sundling ◽  
Y. Li ◽  
N. Huynh ◽  
C. Poulsen ◽  
R. Wilson ◽  
...  
Keyword(s):  
High Resolution ◽  
B Cell ◽  
Binding Site ◽  
Receptor Binding ◽  
Receptor Binding Site ◽  
Cell Responses ◽  
Primary Receptor ◽  
B Cell Responses ◽  
Definition Of

scholarly journals Human erythropoietin-specific sites of monoclonal antibody-mediated neutralization

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 670-675
Author(s):  
MR Fibi ◽  
M Aslan ◽  
P Hintz-Obertreis ◽  
JU Pauly ◽  
M Gerken ◽  
...  
Keyword(s):  
Binding Site ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Enzyme Linked Immunosorbent Assay ◽  
Receptor Binding Site ◽  
Epitope Region ◽  
Human Erythropoietin ◽  
Group A ◽  
Group B ◽  
Binding Determinants

Recombinant human erythropoietin (rhuEpo)-specific mouse monoclonal antibodies (MoAbs) have been produced and characterized. All antibodies were specifically reactive with rhuEpo in enzyme-linked immunosorbent assay (ELISA). Epitope exclusion studies showed three distinct epitope regions, A, B, and C, recognized by neutralizing MoAbs. An additional epitope region D was recognized by non-neutralizing MoAbs. Antibodies defining an epitope region competed with each other for binding sites, but did not compete with antibodies defining a different epitope region. Group B antibodies were able to compete for the receptor binding site on rhuEpo with a soluble human Epo-receptor-lg fusion protein. No single peptide sequences were found to specifically interact either with group B MoAbs or with the rhuEpo-receptor. Therefore, it is suggested that epitope region B and the receptor binding site share binding determinants that are primarily composed of conformational epitopes. Because group A and group C antibodies did not compete with the receptor for binding to the receptor binding site of the rhuEpo molecule, it is suggested that neutralization via epitope regions A and C is mediated through binding inhibition caused by conformational changes, transmuting the binding site(s) for the receptor. Conversely, binding to the receptor seems to induce conformational changes in the hormone molecule, eliminating epitopes for group A and C antibodies.

scholarly journals Human erythropoietin-specific sites of monoclonal antibody-mediated neutralization

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 670-675 ◽  
Author(s):  
MR Fibi ◽  
M Aslan ◽  
P Hintz-Obertreis ◽  
JU Pauly ◽  
M Gerken ◽  
...  
Keyword(s):  
Binding Site ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Enzyme Linked Immunosorbent Assay ◽  
Receptor Binding Site ◽  
Epitope Region ◽  
Human Erythropoietin ◽  
Group A ◽  
Group B ◽  
Binding Determinants

Abstract Recombinant human erythropoietin (rhuEpo)-specific mouse monoclonal antibodies (MoAbs) have been produced and characterized. All antibodies were specifically reactive with rhuEpo in enzyme-linked immunosorbent assay (ELISA). Epitope exclusion studies showed three distinct epitope regions, A, B, and C, recognized by neutralizing MoAbs. An additional epitope region D was recognized by non-neutralizing MoAbs. Antibodies defining an epitope region competed with each other for binding sites, but did not compete with antibodies defining a different epitope region. Group B antibodies were able to compete for the receptor binding site on rhuEpo with a soluble human Epo-receptor-lg fusion protein. No single peptide sequences were found to specifically interact either with group B MoAbs or with the rhuEpo-receptor. Therefore, it is suggested that epitope region B and the receptor binding site share binding determinants that are primarily composed of conformational epitopes. Because group A and group C antibodies did not compete with the receptor for binding to the receptor binding site of the rhuEpo molecule, it is suggested that neutralization via epitope regions A and C is mediated through binding inhibition caused by conformational changes, transmuting the binding site(s) for the receptor. Conversely, binding to the receptor seems to induce conformational changes in the hormone molecule, eliminating epitopes for group A and C antibodies.

scholarly journals Nanobody mediated neutralization reveals an Achilles heel for norovirus

2020 ◽  
Author(s):  
Anna D. Koromyslova ◽  
Jessica Michelle Devant ◽  
Turgay Kilic ◽  
Charles D. Sabin ◽  
Virginie Malak ◽  
...  
Keyword(s):  
Cell Culture ◽  
Binding Site ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Structural Modification ◽  
Cultured Cells ◽  
Murine Norovirus ◽  
Human Norovirus ◽  
Receptor Binding Site ◽  
P Domain

ABSTRACTHuman norovirus frequently causes outbreaks of acute gastroenteritis. Although discovered more than five decades ago, antiviral development has, until recently, been hampered by the lack of a reliable human norovirus cell culture system. Nevertheless, a lot of pathogenesis studies were accomplished using murine norovirus (MNV), which can be grown routinely in cell culture. In this study, we analysed a sizeable library of Nanobodies that were raised against the murine norovirus virion with the main purpose of developing Nanobody-based inhibitors. We discovered two types of neutralizing Nanobodies and analysed the inhibition mechanisms using X-ray crystallography, cryo-EM, and cell culture techniques. The first type bound on the top region of the protruding (P) domain. Interestingly, the Nanobody binding region closely overlapped the MNV receptor-binding site and collectively shared numerous P domain-binding residues. In addition, we showed that these Nanobodies competed with the soluble receptor and this action blocked virion attachment to cultured cells. The second type bound at a dimeric interface on the lower side of the P dimer. We discovered that these Nanobodies disrupted a structural change in the capsid associated with binding co-factors (i.e., metal cations/bile acid). Indeed, we found that capsids underwent major conformational changes following addition of Mg2+ or Ca2+. Ultimately, these Nanobodies directly obstructed a structural modification reserved for a post-receptor attachment stage. Altogether, our new data show that Nanobody-based inhibition could occur by blocking functional and structural capsid properties.AUTHOR SUMMARYThis research discovered and analysed two different types of MNV neutralizing Nanobodies. The top-binding Nanobodies sterically inhibited the receptor-binding site, whereas the dimeric-binding Nanobodies interfered with a structural modification associated with co-factor binding. Moreover, we found that the capsid contained a number of vulnerable regions that were essential for viral replication. In fact, the capsid appeared to be organized in a state of flux, which could be important for co-factor/receptor binding functions. Blocking these capsid-binding events with Nanobodies directly inhibited essential capsid functions. Moreover, a number of MNV-specific Nanobody binding epitopes were comparable to human norovirus-specific Nanobody inhibitors. Therefore, this additional structural and inhibition information could be further exploited in the development of human norovirus antivirals.

scholarly journals Nanobody-Mediated Neutralization Reveals an Achilles Heel for Norovirus

2020 ◽  
Vol 94 (13) ◽  
Author(s):  
Anna D. Koromyslova ◽  
Jessica M. Devant ◽  
Turgay Kilic ◽  
Charles D. Sabin ◽  
Virginie Malak ◽  
...  
Keyword(s):  
Cell Culture ◽  
Binding Site ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Structural Modification ◽  
Cultured Cells ◽  
Murine Norovirus ◽  
Human Norovirus ◽  
Receptor Binding Site ◽  
P Domain

ABSTRACT Human norovirus frequently causes outbreaks of acute gastroenteritis. Although discovered more than five decades ago, antiviral development has, until recently, been hampered by the lack of a reliable human norovirus cell culture system. Nevertheless, a lot of pathogenesis studies were accomplished using murine norovirus (MNV), which can be grown routinely in cell culture. In this study, we analyzed a sizeable library of nanobodies that were raised against the murine norovirus virion with the main purpose of developing nanobody-based inhibitors. We discovered two types of neutralizing nanobodies and analyzed the inhibition mechanisms using X-ray crystallography, cryo-electron microscopy (cryo-EM), and cell culture techniques. The first type bound on the top region of the protruding (P) domain. Interestingly, this nanobody binding region closely overlapped the MNV receptor-binding site and collectively shared numerous P domain-binding residues. In addition, we showed that these nanobodies competed with the soluble receptor, and this action blocked virion attachment to cultured cells. The second type bound at a dimeric interface on the lower side of the P dimer. We discovered that these nanobodies disrupted a structural change in the capsid associated with binding cofactors (i.e., metal cations/bile acid). Indeed, we found that capsids underwent major conformational changes following addition of Mg2+ or Ca2+. Ultimately, these nanobodies directly obstructed a structural modification reserved for a postreceptor attachment stage. Altogether, our new data show that nanobody-based inhibition could occur by blocking functional and structural capsid properties. IMPORTANCE This research discovered and analyzed two different types of MNV-neutralizing nanobodies. The top-binding nanobodies sterically inhibited the receptor-binding site, whereas the dimeric-binding nanobodies interfered with a structural modification associated with cofactor binding. Moreover, we found that the capsid contained a number of vulnerable regions that were essential for viral replication. In fact, the capsid appeared to be organized in a state of flux, which could be important for cofactor/receptor-binding functions. Blocking these capsid-binding events with nanobodies directly inhibited essential capsid functions. Moreover, a number of MNV-specific nanobody binding epitopes were comparable to human norovirus-specific nanobody inhibitors. Therefore, this additional structural and inhibition information could be further exploited in the development of human norovirus antivirals.

Sign in / Sign up

Export Citation Format

Share Document