Box 2.
Non-canonical disulfide linkages of sdAbs
| Some but not all camelid VHHs bear paired cysteine residues, resulting in formation of a second intradomain disulfide linkage in addition to the conserved Cys23-Cys104 linkage (IMGT numbering) present in all Ig domains. Non-canonical disulfide linkages most commonly bridge Cys residues in CDR1 and CDR3,20,21,24 but can also link FR2 and CDR3,21,22 CDR2 and CDR3,23 or two positions within the CDR3 loop.19 The Cys residues in CDR1 are encoded by germline VHH genes that are frequently used in the repertoires of dromedary camels, and B cells using these genes presumably acquire a partner Cys during receptor rearrangement. Two hypotheses have been invoked to explain the presence of non-canonical disulfide linkages in VHH domains: they may impart greater stability to the VHH fold and/or restrict the conformational flexibility of long CDR3 loops, potentially minimizing entropic penalties for antigen binding. However, mutagenesis studies have showed that Cys residues forming non-canonical disulfide linkages can be replaced with a spectrum of other residues with only modest impairment of antigen binding affinity and thermal stability.110 |
| Most cartilaginous fish VNARs bear an additional non-canonical disulfide linkage spanning either FR2-CDR3 (type I) or CDR1-CDR3 (types II and III16). In addition, type I VNARs also bear a CDR3-FR4 disulfide linkage and, sometimes, an intra-CDR3 disulfide linkage (three or four intradomain disulfide linkages in total27). A minority of VNARs (type IV) bear only the single canonical disulfide linkage. As for VHHs, most VNAR Cys residues in CDR1, FR2 and FR4 are probably encoded in the germline and non-canonical disulfide linkages are formed during primary repertoire development4,27,28 |
| Although the precise roles of non-canonical disulfide linkages in sdAb structure and function remain unclear, these linkages very likely influence sdAb paratope structure, since patterns of antigen-driven somatic hypermutation appear to vary depending on their presence and location.27 |