Fig. 4

Functional characterization of the DH domain. a Model for a complex of RhoA with the DH domain of Bcr-Abl. The GTPase binding model was inferred from the co-crystal structure of Dbs and RhoA (PDB ID 1LB1) upon alignment of the Bcr-Abl DH domain with the Dbs DH domain. The α4–α5 loop in Bcr-Abl sterically clashes with RhoA in this model. b Representative SDS-PAGE analysis of the Ni-affinity pull-down experiment using His-tagged DH–PH or DH constructs and RhoA. The prey pull-down efficiency is calculated from signal quantification normalized to the input and shown below the respective lane. I input, FT flow-through, W wash, E elution. c Summary of the pull-down efficiency using either His-tagged DH–PH constructs or GST-tagged GTPases. d Chemical shift perturbation of E510 backbone NH during the NMR titration of the ¹⁵N-labeled DH domain with increasing amounts of RhoA. e Binding affinities for interaction of the three GTPases RhoA, Rac1 and Cdc42 with the DH domain derived from fitting the chemical shift changes with increasing GTPase concentration (Supplementary Fig. 6). Mean K _d values ± s.d. are indicated. f Nucleotide exchange experiment for mant-GDP-loaded RhoA. The decrease of the fluorescence was monitored in the presence of different Bcr-Abl DH–PH constructs and the Dbs DH–PH as a positive control. Experiments were done in duplicates at two different concentrations of the Bcr-Abl DH and DH–PH constructs. g Acceleration of the RhoA nucleotide exchange, i.e., the rate constant in the presence of a DH–PH construct compared to the RhoA intrinsic nucleotide exchange rate, plotted for the different DH–PH constructs at the respective concentrations. h, i Acceleration of Rac1 h and Cdc42 i nucleotide exchange for the different DH–PH constructs at the respective concentrations. Raw data are shown in Supplementary Fig. 8