FcαRI binding at the IgA1 CH2–CH3 interface induces long-range conformational changes that are transmitted to the hinge region

IgA effector functions include proinflammatory immune responses triggered upon clustering of the IgA-specific receptor, FcαRI, by IgA immune complexes. FcαRI binds to the IgA1–Fc domain (Fcα) at the CH2–CH3 junction and, except for CH2 L257 and L258, all side-chain contacts are contributed by the CH3 domain. In this study, we used experimental and computational approaches to elucidate energetic and conformational aspects of FcαRI binding to IgA. The energetic contribution of each IgA residue in the binding interface was assessed by alanine-scanning mutagenesis and equilibrium surface plasmon resonance (SPR). As expected, hydrophobic residues central to the binding site have strong energetic contributions to the FcαRI:Fcα interaction. Surprisingly, individual mutation of CH2 residues L257 and L258, found at the periphery of the FcαRI binding site, dramatically reduced binding affinity. Comparison of antibody:receptor complexes involving IgA or its precursor IgY revealed a conserved receptor binding site at the CH2–CH3 junction (or its equivalent). Given the importance of residues near the CH2–CH3 junction, we used coarse-grained Langevin dynamics simulations to understand the functional dynamics in Fcα. Our simulations indicate that FcαRI binding, either in an asymmetric (1:1) or symmetric (2:1) complex with Fcα, propagated long-range conformational changes across the Fc domains, potentially impacting the hinge and Fab regions. Subsequent SPR experiments confirmed that FcαRI binding to the Fcα CH2–CH3 junction altered the kinetics of HAA lectin binding at the IgA1 hinge. Receptor-induced long-distance conformational transitions have important implications for the interaction of aberrantly glycosylated IgA1 with anti-glycan autoantibodies in IgA nephropathy.

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Identification of the SV2 protein receptor-binding site of botulinum neurotoxin type E

Biochemical Journal ◽

10.1042/bj20130391 ◽

2013 ◽

Vol 453 (1) ◽

pp. 37-47 ◽

Cited By ~ 31

Author(s):

Stefan Mahrhold ◽

Jasmin Strotmeier ◽

Consuelo Garcia-Rodriguez ◽

Jianlong Lou ◽

James D. Marks ◽

...

Keyword(s):

Binding Site ◽

Synaptic Vesicle ◽

Specific Binding ◽

Cell Surface Receptor ◽

Vesicle Membrane ◽

Receptor Binding Site ◽

Binding Interface ◽

Protein Receptor ◽

Surface Receptor ◽

Extended Surface

The highly specific binding and uptake of BoNTs (botulinum neurotoxins; A–G) into peripheral cholinergic motoneurons turns them into the most poisonous substances known. Interaction with gangliosides accumulates the neurotoxins on the plasma membrane and binding to a synaptic vesicle membrane protein leads to neurotoxin endocytosis. SV2 (synaptic vesicle glycoprotein 2) mediates the uptake of BoNT/A and /E, whereas Syt (synaptotagmin) is responsible for the endocytosis of BoNT/B and /G. The Syt-binding site of the former was identified by co-crystallization and mutational analyses. In the present study we report the identification of the SV2-binding interface of BoNT/E. Mutations interfering with SV2 binding were located at a site that corresponds to the Syt-binding site of BoNT/B and at an extended surface area located on the back of the conserved ganglioside-binding site, comprising the N- and C-terminal half of the BoNT/E-binding domain. Mutations impairing the affinity also reduced the neurotoxicity of full-length BoNT/E at mouse phrenic nerve hemidiaphragm preparations demonstrating the crucial role of the identified binding interface. Furthermore, we show that a monoclonal antibody neutralizes BoNT/E activity because it directly interferes with the BoNT/E–SV2 interaction. The results of the present study suggest a novel mode of binding for BoNTs that exploit SV2 as a cell surface receptor.

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Coarse-Grained Models Reveal Functional Dynamics - I. Elastic Network Models – Theories, Comparisons and Perspectives

Bioinformatics and Biology Insights ◽

10.4137/bbi.s460 ◽

2008 ◽

Vol 2 ◽

pp. BBI.S460 ◽

Cited By ~ 21

Author(s):

Lee-Wei Yang ◽

Choon-Peng Chng

Keyword(s):

Conformational Changes ◽

Conformational Dynamics ◽

Computational Cost ◽

Network Models ◽

Coarse Graining ◽

Coarse Grained ◽

Elastic Network ◽

Elastic Network Models ◽

Time Space ◽

Functional Dynamics

In this review, we summarize the progress on coarse-grained elastic network models (CG-ENMs) in the past decade. Theories were formulated to allow study of conformational dynamics in time/space frames of biological interest. Several highlighted models and their underlined hypotheses are introduced in physical depth. Important ENM offshoots, motivated to reproduce experimental data as well as to address the slow-mode-encoded configurational transitions, are also introduced. With the theoretical developments, computational cost is significantly reduced due to simplified potentials and coarse-grained schemes. Accumulating wealth of data suggest that ENMs agree equally well with experiment in describing equilibrium dynamics despite their distinct potentials and levels of coarse-graining. They however do differ in the slowest motional components that are essential to address large conformational changes of functional significance. The difference stems from the dissimilar curvatures of the harmonic energy wells described for each model. We also provide our views on the predictability of ‘open to close’ (open→close) transitions of biomolecules on the basis of conformational selection theory. Lastly, we address the limitations of the ENM formalism which are partially alleviated by the complementary CG-MD approach, to be introduced in the second paper of this two-part series.

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Human erythropoietin-specific sites of monoclonal antibody-mediated neutralization

Blood ◽

10.1182/blood.v81.3.670.bloodjournal813670 ◽

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.

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Human erythropoietin-specific sites of monoclonal antibody-mediated neutralization

Blood ◽

10.1182/blood.v81.3.670.670 ◽

1993 ◽

Vol 81 (3) ◽

pp. 670-675 ◽

Cited By ~ 6

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.

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Nanobody mediated neutralization reveals an Achilles heel for norovirus

10.1101/2020.02.07.938506 ◽

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.

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Molecular modelling studies of a nerve growth factor receptor

Canadian Journal of Chemistry ◽

10.1139/v98-183 ◽

1998 ◽

Vol 76 (10) ◽

pp. 1389-1401 ◽

Cited By ~ 3

Author(s):

Igor L Shamovsky ◽

Gregory M Ross ◽

Richard J Riopelle ◽

Donald F Weaver

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Binding Site ◽

Conformational Changes ◽

Van Der Waals ◽

Protein Docking ◽

Van Der Waals Interactions ◽

Multiple Point ◽

Receptor Binding Site ◽

Nerve Growth

Using computer simulations, a geometry for the receptor binding site for nerve growth factor (NGF) has been proposed. Variable basis Monte Carlo simulated annealing calculations have been used to ascertain the structures of the complexes of four fully active NGF analogues with the second leucine-rich motif (LRM-2) of trkA, a putative binding site for NGF. The previously suggested bioactive conformation of the amino and carboxyl termini of NGF docks favourably with the receptor defined by the LRM-2 of trkA: only minor conformational changes take place in the NGF analogues upon docking. Extensive intermolecular van der Waals contacts arise from the geometric fit of the NGF binding domain to the LRM-2. Within this receptor environment, five distinct binding areas reveal a highly selective multiple-point NGF-trkA recognition based on hydrophobic, ionic, hydrogen bonding, and van der Waals interactions. Binding specificity is determined primarily by residues Lys100, Asp109, and Phe113 of trkA which bind to conserved NGF residues Asp16, Arg114, Lys115, and Phe7. An explicit atom-level model of the high-affinity NGF receptor is thus developed.Key words: NGF, trkA, leucine-rich motif, protein docking, Alzheimer's disease.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2025452118 ◽

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.

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Nanobody-Mediated Neutralization Reveals an Achilles Heel for Norovirus

Journal of Virology ◽

10.1128/jvi.00660-20 ◽

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.

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