Hapten-Induced Dimerization of a Single-Domain VHH Camelid Antibody

Sonneson, Gregory J.; Horn, James R.

doi:10.1021/bi900862r

Cited by 24 publications

(30 citation statements)

References 22 publications

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“…As compared to conventional anti‐hapten antibodies, which primarily use the VH/VL interface to recognize low‐molecular weight ligands, VHH domains would apparently possess a disadvantage in their ability to recognize small molecule haptens. However, biophysical and structural data have revealed that camelid VHH domains can recognize haptens using multiple mechanisms, including using the former VH/VL interface,19, 20 as well as VHH homodimerization 32. Here, structural and energetic studies of the anti‐MTX VHH complex revealed a new hapten binding site, which allows significant MTX penetration and surface area burial.…”

Section: Discussionmentioning

confidence: 92%

An anti‐hapten camelid antibody reveals a cryptic binding site with significant energetic contributions from a nonhypervariable loop

Fanning

Horn

2011

Protein Science

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Conventional anti-hapten antibodies typically bind low-molecular weight compounds (haptens) in the crevice between the variable heavy and light chains. Conversely, heavy chain-only camelid antibodies, which lack a light chain, must rely entirely on a single variable domain to recognize haptens. While several anti-hapten VHHs have been generated, little is known regarding the underlying structural and thermodynamic basis for hapten recognition. Here, an antimethotrexate VHH (anti-MTX VHH) was generated using grafting methods whereby the three complementarity determining regions (CDRs) were inserted onto an existing VHH framework. Thermodynamic analysis of the anti-MTX VHH CDR1-3 Graft revealed a micromolar binding affinity, while the crystal structure of the complex revealed a somewhat surprising noncanonical binding site which involved MTX tunneling under the CDR1 loop. Due to the close proximity of MTX to CDR4, a nonhypervariable loop, the CDR4 loop sequence was subsequently introduced into the CDR1-3 graft, which resulted in a dramatic 1000-fold increase in the binding affinity. Crystal structure analysis of both the free and complex anti-MTX CDR1-4 graft revealed CDR4 plays a significant role in both intermolecular contacts and binding site conformation that appear to contribute toward high affinity binding. Additionally, the anti-MTX VHH possessed relatively high specificity for MTX over closely related compounds aminopterin and folate, demonstrating that VHH domains are capable of binding low-molecular weight ligands with high affinity and specificity, despite their reduced interface.

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Section: Discussionmentioning

confidence: 92%

An anti‐hapten camelid antibody reveals a cryptic binding site with significant energetic contributions from a nonhypervariable loop

Fanning

Horn

2011

Protein Science

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“…Similar binding functionalities may exist for the small ligand caffeine which efficiently mediates the high enthalpy assembly of two V HH domains into a ternary complex of unknown structure. 52 …”

Section: Discussionmentioning

confidence: 99%

Malachite Green Mediates Homodimerization of Antibody VL Domains to Form a Fluorescent Ternary Complex with Singular Symmetric Interfaces

Szent-Györgyi

Stanfield

Andreko

et al. 2013

Journal of Molecular Biology

125

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We report that a symmetric small molecule ligand mediates the assembly of antibody light chain variable domains (VLs) into a correspondent symmetric ternary complex with novel interfaces. The L5* Fluorogen Activating Protein (FAP) is a VL domain that binds malachite green dye (MG) to activate intense fluorescence. Crystallography of liganded L5* reveals a 2:1 protein:ligand complex with inclusive C2 symmetry, where MG is almost entirely encapsulated between an antiparallel arrangement of the two VL domains. Unliganded L5* VL domains crystallize as a similar antiparallel VL/VL homodimer. The complementarity determining regions (CDRs) are spatially oriented to form novel VL/VL and VL/ligand interfaces that tightly constrain a propeller conformer of MG. Binding equilibrium analysis suggests highly cooperative assembly to form a very stable VL/MG/VL complex, such that MG behaves as a strong chemical inducer of dimerization. Fusion of two VL domains into a single protein tightens MG binding over 1,000-fold to low picomolar affinity without altering the large binding enthalpy, suggesting that bonding interactions with ligand and restriction of domain movements make independent contributions to binding. Fluorescence activation of a symmetrical fluorogen provides a selection mechanism for the isolation and directed evolution of ternary complexes where unnatural symmetric binding interfaces are favored over canonical antibody interfaces. As exemplified by L5*, these self-reporting complexes may be useful as modulators of protein association or as high affinity protein tags and capture reagents.

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“…Refolded protein was centrifuged (25,900 g ; 15 min) to remove insoluble precipitate. The refolded VHH protein was subsequently purified as described previously 39…”

Section: Methodsmentioning

confidence: 99%

A combinatorial histidine scanning library approach to engineer highly pH‐dependent protein switches

et al. 2011

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There is growing interest in the development of protein switches, which are proteins whose function, such as binding a target molecule, can be modulated through environmental triggers. Efforts to engineer highly pH sensitive protein-protein interactions typically rely on the rational introduction of ionizable groups in the protein interface. Such experiments are typically time intensive and often sacrifice the protein's affinity at the permissive pH. The underlying thermodynamics of proton-linkage dictate that the presence of multiple ionizable groups, which undergo a pK a change on protein binding, are necessary to result in highly pH-dependent binding. To test this hypothesis, a novel combinatorial histidine library was developed where every possible combination of histidine and wild-type residue is sampled throughout the interface of a model anti-RNase A single domain VHH antibody. Antibodies were coselected for high-affinity binding and pH-sensitivity using an in vitro, dual-function selection strategy. The resulting antibodies retained near wild-type affinity yet became highly sensitive to small decreases in pH, drastically decreasing their binding affinity, due to the incorporation of multiple histidine groups. Several trends were observed, such as histidine ''hot-spots,'' which will help enhance the development of pH switch proteins as well as increase our understanding of the role of ionizable residues in protein interfaces. Overall, the combinatorial approach is rapid, general, and robust and should be capable of producing highly pH-sensitive protein affinity reagents for a number of different applications.

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Hapten-Induced Dimerization of a Single-Domain VHH Camelid Antibody

Cited by 24 publications

References 22 publications

An anti‐hapten camelid antibody reveals a cryptic binding site with significant energetic contributions from a nonhypervariable loop

An anti‐hapten camelid antibody reveals a cryptic binding site with significant energetic contributions from a nonhypervariable loop

Malachite Green Mediates Homodimerization of Antibody VL Domains to Form a Fluorescent Ternary Complex with Singular Symmetric Interfaces

A combinatorial histidine scanning library approach to engineer highly pH‐dependent protein switches

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