Figure 5
From: Structural basis for the inhibition of HIV-1 Nef by a high-affinity binding single-domain antibody
Neffin binding to Nef results in a 2:2 complex formation. (A) Molecular architecture of the fusion of sdAb19 with SH3B6 using a flexible linker of 8, 18, or 38 amino acid length. (B) Analytical gel filtration of Nef with sdAb19 and SH3B6 or Neffin (8 aa linker) reveals a significant size increase for the Nef–Neffin complex. (C) Isothermal titration calorimetry confirms tight binding between Nef and Neffin exhibiting a Kd of 1.6 nM. (D) The triple mutation D60R/G102R/S103E in the sdAb19 subunit increased the dissociation constant of Neffintriple binding to Nef to the portion of the SH3B6 domain alone as determined by ITC measurements. (E) The elution volume of the Nef–Neffintriple complex corresponds to an equimolar 1:1 binding stoichiometry due to the lack of sdAb19-Nef interaction.