title: An anti-hapten camelid antibody reveals a cryptic binding site with significant energetic contributions from a nonhypervariable loop.
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.
authors.affiliation: Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115, USA.
authors.forename: Sean W
authors.initials: SW
authors.lastname: Fanning
authors.forename: James R
authors.initials: JR
authors.lastname: Horn
mesh_terms: D000818:Animals; D002161:Camelids, New World / Q000276:immunology*; D002162:Camelus / Q000276:immunology*; D022801:Complementarity Determining Regions / Q000737:chemistry / Q000276:immunology*; D018360:Crystallography, X-Ray; D006241:Haptens / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000276:immunology*; D007166:Immunosuppressive Agents / Q000276:immunology*; D008727:Methotrexate / Q000276:immunology*; D008958:Models, Molecular; D011487:Protein Conformation; D013816:Thermodynamics
chemical_list: D022801:Complementarity Determining Regions; D006241:Haptens; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D007166:Immunosuppressive Agents; D008727:Methotrexate