Hiding in plain sight: structure and sequence analysis reveals the importance of the antibody DE loop for antibody-antigen binding

Kelow, Simon; Adolf-Bryfogle, Jared; Dunbrack, Roland L.

doi:10.1080/19420862.2020.1840005

Cited by 21 publications

(34 citation statements)

References 81 publications

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“…The thickness of the arrows corresponds to obtained transition times which occur in the micro-to-millisecond timescale. The type of residue of 71 H has already previously been shown to co-determine the canonical structure of the CDR-H2 loop ( 9 ).…”

Section: Resultsmentioning

confidence: 90%

“…In this study we thermodynamically and kinetically characterize the effect of a single point mutation at position 71 H for a human germline IGHV1-69/IGKV1-39 antibody on the paratope states in solution and give a structural and mechanistical explanation of the observed conformational changes. Various studies have already investigated the role of framework mutations on the CDR loops and the relative V H –V L interdomain orientations based on X-ray structures ( 9 , 64 , 65 ). Even allosteric effects involving mutations in the C H 1–C L and the elbow angle have been reported to influence the antibody binding site and consequentially antibody affinity and specificity ( 17 – 22 , 66 – 68 ).…”

Section: Discussionmentioning

confidence: 99%

“…Especially interesting is that the 71 H residue belongs to the Vernier-zone residues, which have been discussed to play a critical role in the humanization and for the rational design of antibodies in general as they can influence antibody specificity and affinity ( 65 , 75 , 76 ). Additionally, residue 71 H is part of the DE loop, also called H4 loop, which joins strands D and E on the heavy chain variable domain ( 9 ). The H4 loop has been traditionally considered to be part of the antibody framework; however, it has been shown not only for antibodies but also for T-cell receptors that the H4 loop can directly interact with the antigen and thus, influence antigen binding ( 9 , 40 ).…”

Section: Discussionmentioning

confidence: 99%

“…Additionally, residue 71 H is part of the DE loop, also called H4 loop, which joins strands D and E on the heavy chain variable domain ( 9 ). The H4 loop has been traditionally considered to be part of the antibody framework; however, it has been shown not only for antibodies but also for T-cell receptors that the H4 loop can directly interact with the antigen and thus, influence antigen binding ( 9 , 40 ). The fact that one single residue in the H4 loop can determine different paratope conformations in solution strongly supports the idea of highly correlated CDR loop movements, which interconvert into each other on the micro-to-millisecond timescale and favor specific interdomain orientations ( 12 , 13 , 41 , 62 , 68 , 77 , 78 ).…”

Section: Discussionmentioning

confidence: 99%

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Mutation of Framework Residue H71 Results in Different Antibody Paratope States in Solution

et al. 2021

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Characterizing and understanding the antibody binding interface have become a pre-requisite for rational antibody design and engineering. The antigen-binding site is formed by six hypervariable loops, known as the complementarity determining regions (CDRs) and by the relative interdomain orientation (VH–VL). Antibody CDR loops with a certain sequence have been thought to be limited to a single static canonical conformation determining their binding properties. However, it has been shown that antibodies exist as ensembles of multiple paratope states, which are defined by a characteristic combination of CDR loop conformations and interdomain orientations. In this study, we thermodynamically and kinetically characterize the prominent role of residue 71H (Chothia nomenclature), which does not only codetermine the canonical conformation of the CDR-H2 loop but also results in changes in conformational diversity and population shifts of the CDR-H1 and CDR-H3 loop. As all CDR loop movements are correlated, conformational rearrangements of the heavy chain CDR loops also induce conformational changes in the CDR-L1, CDR-L2, and CDR-L3 loop. These overall conformational changes of the CDR loops also influence the interface angle distributions, consequentially leading to different paratope states in solution. Thus, the type of residue of 71H, either an alanine or an arginine, not only influences the CDR-H2 loop ensembles, but co-determines the paratope states in solution. Characterization of the functional consequences of mutations of residue 71H on the paratope states and interface orientations has broad implications in the field of antibody engineering.

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Section: Resultsmentioning

confidence: 90%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Mutation of Framework Residue H71 Results in Different Antibody Paratope States in Solution

et al. 2021

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“…The CpHMD simulation results were similar to the GaMD/CpHMD simulation results. Hydrogen bonds play a unique and functionally important role in antibody/antigen interactions [ 53 , 54 , 55 ]. Comparison of W1-Humira binding modes to the wild type Humira showed that there were no hydrogen bonds in W1-Humira/TNF alpha complex stricture at pH 6.0.…”

Section: Discussionmentioning

confidence: 99%

Anti-TNF Alpha Antibody Humira with pH-dependent Binding Characteristics: A constant-pH Molecular Dynamics, Gaussian Accelerated Molecular Dynamics, and In Vitro Study

Hong

Wang

et al. 2021

Biomolecules

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Humira is a monoclonal antibody that binds to TNF alpha, inactivates TNF alpha receptors, and inhibits inflammation. Neonatal Fc receptors can mediate the transcytosis of Humira–TNF alpha complex structures and process them toward degradation pathways, which reduces the therapeutic effect of Humira. Allowing the Humira–TNF alpha complex structures to dissociate to Humira and soluble TNF alpha in the early endosome to enable Humira recycling is crucial. We used the cytoplasmic pH (7.4), the early endosomal pH (6.0), and pKa of histidine side chains (6.0–6.4) to mutate the residues of complementarity-determining regions with histidine. Our engineered Humira (W1-Humira) can bind to TNF alpha in plasma at neutral pH and dissociate from the TNF alpha in the endosome at acidic pH. We used the constant-pH molecular dynamics, Gaussian accelerated molecular dynamics, two-dimensional potential mean force profiles, and in vitro methods to investigate the characteristics of W1-Humira. Our results revealed that the proposed Humira can bind TNF alpha with pH-dependent affinity in vitro. The W1-Humira was weaker than wild-type Humira at neutral pH in vitro, and our prediction results were close to the in vitro results. Furthermore, our approach displayed a high accuracy in antibody pH-dependent binding characteristics prediction, which may facilitate antibody drug design. Advancements in computational methods and computing power may further aid in addressing the challenges in antibody drug design.

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Arginine cluster introduction on framework region in anti‐lysozyme antibody improved association rate constant by changing conformational diversity of CDR loops

et al. 2023

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Antibodies are used for many therapeutic and biotechnological purposes. Because the affinity of an antibody to the antigen is critical for clinical efficacy of pharmaceuticals, many affinity maturation strategies have been developed. Although we previously reported an affinity maturation strategy in which the association rate of the antibody towards its antigen is improved by introducing a cluster of arginine residues into the framework region of the antibody, the detailed molecular mechanism responsible for this improvement has been unknown. In this study, we introduced five arginine residues into an anti‐hen egg white lysozyme antibody (HyHEL10) Fab fragment to create the R5‐mutant and comprehensively characterized the interaction between antibody and antigen using thermodynamic analysis, X‐ray crystallography, and molecular dynamics (MD) simulations. Our results indicate that introduction of charged residues strongly enhanced the association rate, as previously reported, and the antibody‐antigen complex structure was almost the same for the R5‐mutant and wild‐type Fabs. The MD simulations indicate that the mutation increased conformational diversity in complementarity‐determining region loops and thereby enhanced the association rate. These observations provide the molecular basis of affinity maturation by R5 mutation.This article is protected by copyright. All rights reserved.

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Hiding in plain sight: structure and sequence analysis reveals the importance of the antibody DE loop for antibody-antigen binding

Cited by 21 publications

References 81 publications

Mutation of Framework Residue H71 Results in Different Antibody Paratope States in Solution

Mutation of Framework Residue H71 Results in Different Antibody Paratope States in Solution

Anti-TNF Alpha Antibody Humira with pH-dependent Binding Characteristics: A constant-pH Molecular Dynamics, Gaussian Accelerated Molecular Dynamics, and In Vitro Study

Arginine cluster introduction on framework region in anti‐lysozyme antibody improved association rate constant by changing conformational diversity of CDR loops

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