Functional insights of Tyr37 in framework region 2 directly contributing to the binding affinities and dissociation kinetics in single-domain VHH antibodies

• Published in: Biochemical and biophysical research communications 2024-05, Vol.709, p.149839, Article 149839
• Main Authors: Yamamoto, Koichi, Nagatoishi, Satoru, Nakakido, Makoto, Kuroda, Daisuke, Tsumoto, Kouhei
• Format: Article
• Language: English
• Subjects: Binding affinity; Framework region 2; Kinetics; Position 37; Tyrosine; VHH antibody
• ISSN: 0006-291X; 1090-2104; 1090-2104
• DOI: 10.1016/j.bbrc.2024.149839

Single-domain VHH antibody is regarded as one of the promising antibody classes for therapeutic and diagnostic applications. VHH antibodies have amino acids in framework region 2 that are distinct from those in conventional antibodies, such as the Val37Phe/Tyr (V37F/Y) substitution. Correlations between the residue type at position 37 and the conformation of the CDR3 in VHH antigen recognition have been previously reported. However, few studies focused on the meaning of harboring two residue types in position 37 of VHH antibodies, and the concrete roles of Y37 have been little to be elucidated. Here, we investigated the functional states of position 37 in co-crystal structures and performed analyses of three model antibodies with either F or Y at position 37. Our analysis indicates that Y at position 37 enhances the dissociation rate, which is highly correlated with drug efficacy. Our findings help to explain the molecular mechanisms that distinguish VHH antibodies from conventional antibodies. •VHH antibodies with β-hairpin CDR3 have a characteristic tyrosine residue at position 37 in the FR2 region.•Functional roles of Y37 were investigated based on the complex structures and Y37F mutational analysis.•Direct interaction of Y37 was effective for enhancing the dissociation rate constant, which is largely related to drug efficacy.•The position 37 residue must be functionally considered in the sequence design and optimization of VHH antibodies with short extended CDR3 regions.