Preliminary crystallographic study of a complex between the Fab fragment of a monoclonal anti-lysozyme antibody (D1.3) and the Fab fragment from an anti-idiotopic antibody against D1.3

Boulot, Ginnette; Rojas, Carlos Antônio Aguirre; Bentley, G.A.; Poljak, Roberto J.; Barbier, Eliane; Guern, Christian Le; Cazenave, Pierre‐André

doi:10.1016/0022-2836(87)90685-1

Cited by 19 publications

(4 citation statements)

References 8 publications

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“…This panel included recombinant IgG with Fab with a VHIII region, which displayed the expected Fab-mediated binding reactivity with native recombinant SpA (Supplementary Figure 1C ). While those with a defined VHI-Fab did not interact with SpA via this domain ( 52 ) (Supplementary Figure 2A ). These IgG were generated as whole molecules, with murine gamma constant regions of different subclasses, which varied in their capacity for Fc-mediated SpA binding interactions, as anticipated (Supplementary Table 1 , Supplementary Figure 2B ).…”

Section: Resultsmentioning

confidence: 99%

“…Recombinant murine VHIII IgG1, IgG2a or IgG3 were generated with the variable regions of the anti-PC TEPC15 parental clone ( 51 ), while the VHI isotype control IgG express antibody genes from the anti-Hen Egg Lysozyme (HEL) D1.3 mAb ( 52 ), which were generated by transfection of CHO cells and subsequent cloning and expansion, using the OptiVEC system (Invitrogen, Thermo Fischer). Antibody gene sequences of constructs were confirmed by automated sequencing (not shown).…”

Section: Methodsmentioning

confidence: 99%

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Essential Domain-Dependent Roles Within Soluble IgG for in vivo Superantigen Properties of Staphylococcal Protein A: Resolving the B-Cell Superantigen Paradox

2018

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Staphylococcus aureus is a common commensal and frequent opportunistic pathogen that causes invasive infections that often recur. Co-evolution with the host has led to the development of toxins that affect diverse immune cell types. Recent reports have highlighted the contributions of staphylococcal protein A (SpA). This small oligomeric secreted protein contains 4–5 homologous domains with two distinct immunoglobulin-binding sites; one for IgG Fc domains, while a separate site binds an evolutionarily conserved surface on Fab encoded by VHIII clan related genes. The Fab-binding site has been implicated in in vivo supraclonal VHIII-BCR targeted B-cell depletion by an activation induced death pathway. Yet the concept of a superantigen for B lymphocytes poses a seeming paradox. Unlike TCR that are expressed only in a membrane-associated form, BCR are expressed in both a membrane BCR form and in secreted Ig forms, which permeate virtually every part of the body at high levels. We therefore asked, why circulating immunoglobulin do not block the superantigen properties of SpA? Herein, we show that soluble IgG molecules are not in vivo inhibitors of these B-cell superantigen effects but are instead essential for potentiating these properties. We also show that the Fc subclass of circulating IgG is an indirect critical determinant of the B-cell superantigen effect. In contrast, host FcγR and complement are not required for SpA mediated in vivo B-cell depletion. Unexpectedly, after VHIII-IgG2a pretreatment SpA challenge resulted in fatal anaphylactic reactions, which we speculate may have involved FcγR interactions with mast cells and basophils. Cumulatively, our findings illuminate a cunning and potent molecular strategy by which a bacterial toxin effectively confounds the contributions of host B-lymphocytes to immune defenses.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Essential Domain-Dependent Roles Within Soluble IgG for in vivo Superantigen Properties of Staphylococcal Protein A: Resolving the B-Cell Superantigen Paradox

2018

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“…Very importantly, the presence of just a single mutation at the binding interface of the Fv, combined with structural information describing both beforeand after-mutation systems, allows us to disentangle the effects of binding affinity estimation from those of side-chain reconstruction, an aspect which has remained elusive until now. The selected Fvs are derived from X-ray-determined structures of either HyHEL-10 (L 1−107 and H 1−114) 40 or D1.3 Fv (L 1−107 and H 1−116) 41 in complex with HEWL, as exemplified in Figures 1a and S1. Notably, these systems have been reported in a series of articles in which either the 1C08 or the 1VFB structures were used as a wild-type (WT) reference.…”

Section: Molecular Dynamicsmentioning

confidence: 99%

Computational Mutagenesis of Antibody Fragments: Disentangling Side Chains from ΔΔG Predictions

Tandiana,

Barletta,

Soler

et al. 2024

J. Chem. Theory Comput.

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The development of highly potent antibodies and antibody fragments as binding agents holds significant implications in fields such as biosensing and biotherapeutics. Their binding strength is intricately linked to the arrangement and composition of residues at the binding interface. Computational techniques offer a robust means to predict the three-dimensional structure of these complexes and to assess the affinity changes resulting from mutations. Given the interdependence of structure and affinity prediction, our objective here is to disentangle their roles. We aim to evaluate independently six side-chain reconstruction methods and ten binding affinity estimation techniques. This evaluation was pivotal in predicting affinity alterations due to single mutations, a key step in computational affinity maturation protocols. Our analysis focuses on a data set comprising 27 distinct antibody/hen egg white lysozyme complexes, each with crystal structures and experimentally determined binding affinities. Using six different side-chain reconstruction methods, we transformed each structure into its corresponding mutant via in silico single-point mutations. Subsequently, these structures undergo minimization and molecular dynamics simulation. We therefore estimate ΔΔG values based on the original crystal structure, its energy-minimized form, and the ensuing molecular dynamics trajectories. Our research underscores the critical importance of selecting reliable side-chain reconstruction methods and conducting thorough molecular dynamics simulations to accurately predict the impact of mutations. In summary, our study demonstrates that the integration of conformational sampling and scoring is a potent approach to precisely characterizing mutation processes in single-point mutagenesis protocols and crucial for computational antibody design.

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“…E225 is a monoclonal antibody (IgG2b, K) that recognizes an idiotope on the monoclonal anti-lysozyme antibody D1.3 (IgG1, K) (Amit, Mariuzza, Phillips & Poljak, 1986). The complex, FabD1.3-FabE225, formed between the antigen-binding fragments of these two antibodies, crystallizes in the space group P21 (a = 75.1, b=77.7, c=96.8A, /3=111.8 ° ) with one molecule of complex (2 Fabs) in the asymmetric unit (Boulot et aL, 1987). Thus, four independent components must be oriented and positioned in the asymmetric unit: two dimers of each kind.…”

Section: Introduction To Structures Usedmentioning

confidence: 99%

Some applications of the phased translation function in macromolecular structure determination

Bentley

Houdusse²

1992

Acta Cryst Sect A

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Although the phased translation function was first described some time ago [Colman, Fehlhammer & Bartels (1976). In Crystallographic Computing Techniques, edited by F. R. Ahmed, K. Huml & B. Sedhi~ek,, it has been little used, especially in the application of molecular replacement to macromolocular structures. Nevertheless, the procedure is relatively easy to apply and deserves wider use. In this paper the versatility of the phased translation function in a number of different applications is examined and experience gained in obtaining optimal results in protein structure determination by this method is reported. Examples given show how it can be used to position an oriented fragment, to locate independent components with respect to a common crystallographic origin and to choose correctly between enantiomorphic space groups. Its performance is compared with other translation functions in common use.

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Preliminary crystallographic study of a complex between the Fab fragment of a monoclonal anti-lysozyme antibody (D1.3) and the Fab fragment from an anti-idiotopic antibody against D1.3

Cited by 19 publications

References 8 publications

Essential Domain-Dependent Roles Within Soluble IgG for in vivo Superantigen Properties of Staphylococcal Protein A: Resolving the B-Cell Superantigen Paradox

Essential Domain-Dependent Roles Within Soluble IgG for in vivo Superantigen Properties of Staphylococcal Protein A: Resolving the B-Cell Superantigen Paradox

Computational Mutagenesis of Antibody Fragments: Disentangling Side Chains from ΔΔG Predictions

Some applications of the phased translation function in macromolecular structure determination

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