Fig. 3: Zn site and conformation of Nf1 and SPRED1 binding.

Colour scheme as in Fig. 2. a, A conformational state with both protomers in the open conformation was not observed and is impeded by severe steric interference (indicated by the asterick) between the two respective GRDs. b, Insert: density around the metal binding site, with tetrahedral His₂Cys-water coordination and distances typical for Zn²⁺. Right panel: Nf1-23a accelerates the rate of GTP hydrolysis by KRas. Error bars represent the mean ± s.e.m. of n = 5 or n = 8 independent assays of the same sample as indicated in each bar by individual data points. Significance calculated by one-way ANOVA followed by pairwise two-sided t-tests, applying Bonferroni correction with α = 0.01. *P = 0.0016, ***P < 0.000052; NS, not significant. c, Top: concomitant SPRED1 and Ras binding is possible only to the protomer in open conformation. Bottom: In the closed conformation, SPRED1 can bind, whereas Ras cannot bind. d, Superposition of all available GRD and Sec14-PH domains onto the Protein Data Bank (PDB) model 1nf1. The backbone traces of GRD X-ray models are shown light blue, and the X-ray Sec14-PH domains are shown in yellow. The GRD loop C1486-D1490, not visible in the X-ray structures, must undergo rearrangement upon SPRED1 binding in both conformations because it interferes with bound SPRED1.