Structure and specificity of several triclocarban‐binding single domain camelid antibody fragments

Abstract

The variable VHH domains of camelid single chain antibodies have been useful in numerous biotechnology applications due to their simplicity, biophysical properties, and abilities to bind to their cognate antigens with high affinities and specificity. Their interactions with proteins have been well-studied, but considerably less work has been done to characterize their ability to bind haptens. A high-resolution structural study of three nanobodies (T4, T9, and T10) which have been shown to bind triclocarban (TCC, 3-(4-chlorophenyl)-1-(3,4-dichlorophenyl)urea) with near-nanomolar affinity shows that binding occurs in a tunnel largely formed by CDR1 rather than a surface or lateral binding mode seen in other nanobody-hapten interactions. Additional significant interactions are formed with a non-hypervariable loop, sometimes dubbed "CDR4". A comparison of apo and holo forms of T9 and T10 shows that the binding site undergoes little conformational change upon binding of TCC. Structures of three nanobody-TCC complexes demonstrated there was not a standard binding mode. T4 and T9 have a high degree of sequence identity and bind the hapten in a nearly identical manner, while the more divergent T10 binds TCC in a slightly displaced orientation with the urea moiety rotated approximately 180° along the long axis of the molecule. In addition to methotrexate, this is the second report of haptens binding in a tunnel formed by CDR1, suggesting that compounds with similar hydrophobicity and shape could be recognized by nanobodies in analogous fashion. Structure-guided mutations failed to improve binding affinity for T4 and T9 underscoring the high degree of natural optimization.

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

“…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.…”

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

“…RR6 and RR1 are significantly larger than the other two haptens, whereas the binding modes of VHH antibody to RR6 and RR1 are similar to that of traditional IgG antibody, which utilizes all of its CDRs. The molecular weights of the other two haptens are significantly lower, MTX is 454.44 Da [12] and TCC is 315.58 Da [13], and they are more similar to the cortisol (362.46 Da) that we tested in the present study. By comparing the crystal structures of the three available VHH antibody-small hapten (molecular weight < 500 Da) complexes, we found that they all adopt a very special binding mode: the most important CDR3 region in other VHH antibody interactions was barely involved in the interaction with haptens and their binding to hapten was mainly dependent on the CDR1 region.…”

“…Although many structures of complexes involving VHH antibody and the corresponding antigen have been documented, only four types of structure for VHH antibody bound to small molecule haptens, namely azo-dye RR6 [10], RR1 [11], MTX [12] and TCC [13], have been deposited in the PDB to date. RR6 and RR1 are significantly larger than the other two haptens, whereas the binding modes of VHH antibody to RR6 and RR1 are similar to that of traditional IgG antibody, which utilizes all of its CDRs.…”

“…application of VHH antibodies as a probe for smaller molecules remains difficult, especially with the lack of structural information of VHH antibodies. Only few classes of VHH antibodies that bind to haptens have been reported to date: Spinelli et al [10,11] determined the complex structure of azo-dye bound by VHH antibody; Fanning et al [12] determined the structure of nanobodies bound to methotrexate (MTX); and Rosa et al [13] determined the structure of nanobodies bound to triclocarban (TCC).…”