Fig. 4: Structural characteristics of optimized sdAb.

a Structural analysis of key binding sites between human-derived sdAb and Protein A. The 72–76 amino acid residues are highlighted in red, with surrounding β-strands numbered according to previous reports (upper panel). The 74th site mutation to serine (green) or asparagine (cyan) causes steric hindrance with domain D, marked by red circles (lower panel). Amino acid numbering is based on the IMGT nomenclature. b Structural analysis of key binding sites between camel-derived VHH and Protein A. In the upper panel, the interaction between glutamine at position 90 and residues N92 and R20, as well as the interaction between R20 and domain D, is depicted. In the lower panel, the potential interactions between leucine at position 90 and residues N92 and R20, as well as the potential interaction between R20 and domain D, are illustrated. Hydrogen bonds are represented by yellow dashed lines, and salt bridges are indicated by red dashed lines. c The crystal structure of VHH3-M1 and domain D of Protein A (1.49 Å), with VHH3-M1 represented in pink and domain D in light blue. The three CDRs are marked in orange or red, respectively. d The polar interactions between VHH3-M1 and domain D. All interactive residues are shown in stick and colored in pink for VHH3-M1 and light blue for domain D. The hydrogen bonds are shown in black dashes while salt bridge in red. e The comparison of the complex structures between n501, VHH3-M1, and VHH3-M3 with domain D is depicted. Domain D is represented in light blue. n501, VHH3-M1, and VHH3-M3 are displayed in pale green, pink, and orange, respectively. f The comparison of the binding of VHH3-M1 and n501 to domain D is illustrated. The interacting amino acids are displayed as sticks. Pink dashed lines represent the hydrogen bond interactions between VHH3-M1 and domain D, while green dashed lines indicate the hydrogen bond interactions between n501 and domain D. g The comparison of the binding of VHH3-M1 and VHH3-M3 to domain D is presented. The interacting amino acids are depicted as sticks, with residues in VHH3-M1 represented in pink and those in VHH3-M3 in orange. Domain D is depicted in light blue.