title: Dual beneficial effect of interloop disulfide bond for single domain antibody fragments.
abstract: The antigen-binding fragment of functional heavy chain antibodies (HCAbs) in camelids comprises a single domain, named the variable domain of heavy chain of HCAbs (VHH). The VHH harbors remarkable amino acid substitutions in the framework region-2 to generate an antigen-binding domain that functions in the absence of a light chain partner. The substitutions provide a more hydrophilic, hence more soluble, character to the VHH but decrease the intrinsic stability of the domain. Here we investigate the functional role of an additional hallmark of dromedary VHHs, i.e. the extra disulfide bond between the first and third antigen-binding loops. After substituting the cysteines forming this interloop cystine by all 20 amino acids, we selected and characterized several VHHs that retain antigen binding capacity. Although VHH domains can function in the absence of an interloop disulfide bond, we demonstrate that its presence constitutes a net advantage. First, the disulfide bond stabilizes the domain and counteracts the destabilization by the framework region-2 hallmark amino acids. Second, the disulfide bond rigidifies the long third antigen-binding loop, leading to a stronger antigen interaction. This dual beneficial effect explains the in vivo antibody maturation process favoring VHH domains with an interloop disulfide bond.
authors: Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium.
authors: Jochen
authors: J
authors: Govaert
authors: Mireille
authors: M
authors: Pellis
authors: Nick
authors: N
authors: Deschacht
authors: Cécile
authors: C
authors: Vincke
authors: Katja
authors: K
authors: Conrath
authors: Serge
authors: S
authors: Muyldermans
authors: Dirk
authors: D
authors: Saerens
mesh_terms: D000818:Animals; D002162:Camelus; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D004220:Disulfides; D007143:Immunoglobulin Heavy Chains; D057127:Single-Chain Antibodies; D003545:Cysteine