Fig. 2: Competitive binders to SOS1 (Nb77) or RAS (Nb84) inhibit nucleotide exchange, while allosteric binders to the SOS1•RAS complex (Nb14 and Nb22) increase nucleotide exchange.

a Crystal structures of the Nbs (cyan, cartoon representation) in complex with SOS1 (orange) and/or RAS (green). The structure of the ternary complex SOS1•RAS•RAS^Y64A (RAS^Y64A•GppNHp, cartoon representation) is also shown for comparison (pdb ID 1NVV). b SOS1-catalyzed nucleotide exchange assays on RAS performed in presence of the Nbs or RAS^Y64A (black curves). Single turnover nucleotide exchange experiments were started by mixing RAS•mGDP (1 µM) with an excess of GTP (200 µM) at a fixed concentration of SOS1 (0.05, 0.3 or 1 µM depending on the allosteric binder). The red arrows follow increasing concentrations of the Nbs or RAS^Y64A. As references, we included the intrinsic nucleotide exchange of RAS alone (green curves) and the nucleotide exchange of RAS in complex with SOS1 (orange curves). c Single turnover time traces were fitted as single exponential decays and the apparent rates were normalized to the SOS1-catalyzed nucleotide exchange rate (orange curves) and plotted as a function of the nanobody or RAS^Y64A concentration. Data are presented as mean values +/− SEM. Nb77 fully inhibits SOS1 activity (SOS1 at 1 µM, EC50 of 0.95 µM ± 0.1 µM). Nb84 fully inhibits SOS1 activity and abolishes the intrinsic nucleotide exchange reaction of RAS (SOS1 at 1 µM, EC50 of 0.20 ± 0.02 µM). Nb14 increases SOS1 activity 27-fold (SOS1 at 0.05 µM, EC50 of 0.23 µM ± 0.03 µM). Nb22 increases SOS1 activity 2.6-fold (SOS1 at 1 µM, EC50 of 2.0 µM ± 0.3 µM). The allosteric RAS increases SOS1 activity 14-fold (SOS1 at 0.3 µM, EC50 of 6.7 ± 0.5 µM). The experiments were repeated independently three times. Source data are provided as a Source Data file.