3QXW

Free structure of an anti-methotrexate CDR1-4 Graft VHH Antibody

Summary for 3QXW

• Entry DOI: 10.2210/pdb3qxw/pdb
• Related: 1I3U 2x6m 3QXT 3QXU 3QXV
• Descriptor: Anti-Methotrexate CDR1-4 Graft VHH, SODIUM ION, SULFATE ION, ... (4 entities in total)
• Functional Keywords: camelid single domain antibody, heavy chain only, vhh, antibody, anti-hapten antibody, cdr, hapten binding, small molecule sensing, ligand binding, low molecular weight compound, methotrexate, immune system
• Biological source: Lama Glama (Llama)
• Total number of polymer chains: 5
• Total formula weight: 69025.94

Authors

Fanning, S.W.,Horn, J.R. (deposition date: 2011-03-02, release date: 2011-07-27, Last modification date: 2024-11-20)

Primary citation

Fanning, S.W.,Horn, J.R.
An anti-hapten camelid antibody reveals a cryptic binding site with significant energetic contributions from a nonhypervariable loop.
Protein Sci., 20:1196-1207, 2011

PubMed Abstract: 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.

PubMed: 21557375
DOI: 10.1002/pro.648

Experimental method

X-RAY DIFFRACTION (1.85 Å)