Structural insights into the mechanism of single domain VHH antibody binding to cortisol

Abstract

To date, few structural models of VHH antibody binding to low molecular weight haptens have been reported. Here, we report the crystal structure of cortisol binding to its VHH antibody NbCor at pH 3.5 and 10.5. Cortisol binds to NbCor mainly by burying itself under the tunnel formed by the complementarity determining region 1 (CDR1) of NbCor. The affinity of NbCor binding to cortisol and similar compounds was also verified by a microscale thermophoresis assay. Combining our findings with several previously reported structures of hapten‐VHH antibody complexes, we propose that VHH antibodies exhibit a special mechanism of binding small haptens by encapsulating them in a tunnel formed by CDR1. Our findings provide useful structural information for the further development and optimization of hapten‐specific VHH antibodies.

Role of the non‐hypervariable FR3 D‐E loop in single‐domain antibody recognition of haptens and carbohydrates

“…We docked a generic model GlcNAc disaccharide (representing, for instance, the approximate size of terminal sugars of branched N ‐linked oligosaccharides) in the inter‐CDR pocket of cAbAn33. This ligand pose was chosen to be similar to those adopted by low‐molecular weight antigens in complex with other sdAbs (eg, methotrexate, PDB ID: 3QXV; triclocarban, PDB ID: 5VL2; cortisol, PDB IDs: 6ITP and 6ITQ) (Figure S3). In this “CDR1‐tunneling” binding mode, the disaccharide would make minimal contact with CDR3 and would instead draw additional contacts with CDR4.…”

“…The structures of the chemotherapeutic methotrexate, the pesticide triclocarban, and the steroid hormone cortisol in complex with specific sdAbs all pointed to a significant role of CDR4 in hapten recognition and the formation of a binding tunnel involving CDR1 and to a lesser extent CDR3. However, recognition of chemical haptens required nearly complete burial of these molecules in hydrophobic pockets with a significant energetic contribution of FR main‐chain contacts.…”

“…Multiple studies have illustrated that camelid heavy chain‐only antibody responses against haptens are generally weaker than their conventional antibody counterparts . However, only a handful of studies have reported X‐ray crystal structures of camelid sdAbs in complex with haptens or short peptides . In a 2011 report, Fanning et al demonstrated that binding of a methotrexate‐specific sdAb involved a significant energetic contribution of a “cryptic” binding site formed from a non‐hypervariable loop connecting β‐strands D and E of framework region (FR) 3 .…”

“…This loop is sometimes called complementarity‐determining region (CDR)4 and has been previously shown to be involved in binding by an anti‐vascular endothelial growth factor Fab . Binding modes similar to the methotrexate‐specific sdAb of Fanning et al were recently described by Tabares‐da Rosa et al for three camelid sdAbs against triclocarban and by Ding et al for a camelid sdAb against cortisol . For all five sdAbs, the hapten was bound in a “tunnel” under the CDR1 loop and made significant contact with main‐chain atoms.…”

Structure and development of single domain antibodies as modules for therapeutics and diagnostics

“…In addition to intermolecular interactions between methotrexate and both CDR1 and VHH framework residues, "CDR4," the non-hypervariable loop that is near the three CDRs, makes important energetic contributions towards methotrexate binding. Interestingly, structures of three different VHH domains in complex with triclocarban, 54 an antibacterial/antifungal agent, as well as structures of an anti-cortisol VHH complex, 55 revealed that the haptens bound the same tunnel beneath CDR1 as found in the anti-methotrexate VHH. These results suggest that the CDR1 "tunnel" may be a common mechanism for VHH interactions and may serve as a template for designing novel anti-hapten VHH domains.…”