Journal: The EMBO Journal
Article Title: K128 ubiquitination constrains RAS activity by expanding its binding interface with GAP proteins
doi: 10.1038/s44318-024-00146-w
Figure Lengend Snippet: ( A ) Superimposition of five representative complex conformations from the ensemble clusters for the K128-ubiquitinated NRAS/ RASA1 GAP complex. ( B ) The interaction energies of RASA1 GAP with non-ubiquitinated NRAS and NRAS ubiquitinated at K128. The interaction energies were calculated over the entire trajectories and then averaged. Data represented as mean ± SD. N = 1250 snapshots. ( C ) Chemical ubiquitination of NRAS-C118S/ K128C mutant and purification of monoubiquitinated NRAS by size-exclusion chromatography. Proteins were separated by SDS-PAGE under non–reducing conditions or in the presence of a reducing agent, tris(2-carboxyethyl) phosphine (TCEP), and stained by Coomassie blue. ( D ) In vitro Co-IP of non-conjugated or ubiquitin-conjugated NRAS-K128C and RAF1. Equimolar amounts of NRAS-K128C/ Ubiquitin-G76C complex or NRAS-K128C mutant were incubated with V5-tagged RAF1 in the presence of GDP or GTPγS. ( E ) In vitro Co-IP of non-conjugated or ubiquitin-conjugated NRAS-K128C and SOS1. Equimolar amounts of NRAS-K128C-Ubiquitin-G76C complex or NRAS-K128C mutant were incubated with equal amounts of the GDP/GTP exchange domain of SOS1 in the presence of GDP. ( F ) In vitro Co-IP of non-conjugated or ubiquitin-conjugated NRAS-K128C and RASA1. Equimolar amounts of the indicated NRAS proteins were incubated with equal amounts of RASA1 GAP in the presence of GTPγS. ( G ) RASA1-mediated GTPase activity of ubiquitinated and non-ubiquitinated NRAS normalized to intrinsic GTPase activity. Equimolar amounts of the indicated NRAS proteins were mixed with the GAP domain of RASA1. GTP hydrolysis reaction was initiated by the addition of GTP. Data were present as mean ± s.e.m. N = 3 technical replicates in independent experiments. P -value was calculated by a two-sided t -test. ( H ) wt-RAS and ubiquitination-deficient RAS mutant were co-immunoprecipitated with GST-tagged RASA1 GAP using anti-Flag resin followed by immunoblotting with the indicated antibodies. ( I ) Superimposition of five representative complex conformations from the ensemble clusters for the K128-ubiquitinated KRAS/ NF1 GRD complex. ( J ) The interaction energies of NF1 GRD with non-ubiquitinated KRAS and KRAS ubiquitinated at K128. Data represented as mean ± SD. N = 1250 snapshots. ( K ) The indicated RAS proteins were co-immunoprecipitated with GST-tagged NF1 GRD using anti-Flag resin, followed by immunoblotting with the indicated antibodies. .
Article Snippet: The recombinant proteins used: RASA1 Human Recombinant Protein (Full-length, Abnova, P01, NP_072179.1) and the GDP/GTP exchange domain of SOS1 (564-1049aa, Cytoskeleton).
Techniques: Mutagenesis, Purification, Size-exclusion Chromatography, SDS Page, Staining, In Vitro, Co-Immunoprecipitation Assay, Incubation, Activity Assay, Immunoprecipitation, Western Blot
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