Multimerization of HIV-1 integrase hinges on conserved SH3-docking platforms

Meytal Galilee; Akram Alian

Analyses of interactions were performed using COCOMAPS (37) and LigPlot + (38) . Lysine Crosslinking: Homobifunctional BS 3 (Pierce, 2.5 mM) was added to full-length IN (20 µM) that was initially mixed with increasing concentrations of inhibitory peptide or Y3molecule (4-Acetamido-5-hydroxy-2,7-naphthalenedisulfonic acid, (Sigma, S981680)). Reactions were incubated for 30 minutes at room temperature, and quenched by adding Tris-HCl (pH 7.5) to a final concentration of 10 mM and analyzed on SDS-PAGE. Affinity Measurements: IN proteins were labeled with Monolith NT protein labeling kit BLUE-NHS (NanoTemper Technologies) and eluted in microscale thermophoresis (MST) buffer (50 mM Tris-HCl pH 7.0, 300 mM NaCl and 0.05% Tween-20). Labeled IN proteins were incubated with serial dilutions of inhibitor peptide, and MST measurements were carried out using the Monolith NT.115 in standard capillaries with 40% LED and 20% MST power.