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An anti‐hapten camelid antibody reveals a cryptic binding site with significant energetic contributions from a nonhypervariable loop
Author(s) -
Fanning Sean W.,
Horn James R.
Publication year - 2011
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.648
Subject(s) - hapten , chemistry , aminopterin , single domain antibody , complementarity determining region , stereochemistry , binding site , immunoglobulin light chain , biophysics , antibody , biochemistry , peptide sequence , biology , methotrexate , genetics , immunology , gene
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 anti‐methotrexate 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.