6NTC

Crystal Structure of G12V HRas-GppNHp bound in complex with the engineered RBD variant 1 of CRAF Kinase protein

Summary for 6NTC

• Entry DOI: 10.2210/pdb6ntc/pdb
• Descriptor: GTPase HRas, RAF proto-oncogene serine/threonine-protein kinase, PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER, ... (6 entities in total)
• Functional Keywords: affinity reagent, ras inhibitor, transferase-protein binding complex, transferase/protein binding
• Biological source: Homo sapiens (Human); More
• Total number of polymer chains: 2
• Total formula weight: 29191.93

Authors

Maisonneuve, P.,Kurinov, I.,Wiechmann, S.,Ernst, A.,Sicheri, F. (deposition date: 2019-01-28, release date: 2020-03-04, Last modification date: 2023-10-11)

Primary citation

Wiechmann, S.,Maisonneuve, P.,Grebbin, B.M.,Hoffmeister, M.,Kaulich, M.,Clevers, H.,Rajalingam, K.,Kurinov, I.,Farin, H.F.,Sicheri, F.,Ernst, A.
Conformation-specific inhibitors of activated Ras GTPases reveal limited Ras dependency of patient-derived cancer organoids.
J.Biol.Chem., 295:4526-4540, 2020

PubMed Abstract: The small GTPases H, K, and NRAS are molecular switches indispensable for proper regulation of cellular proliferation and growth. Several mutations in the genes encoding members of this protein family are associated with cancer and result in aberrant activation of signaling processes caused by a deregulated recruitment of downstream effector proteins. In this study, we engineered variants of the Ras-binding domain (RBD) of the C-Raf proto-oncogene, Ser/Thr kinase (CRAF). These variants bound with high affinity with the effector-binding site of Ras in an active conformation. Structural characterization disclosed how the newly identified RBD mutations cooperate and thereby enhance affinity with the effector-binding site in Ras compared with WT RBD. The engineered RBD variants closely mimicked the interaction mode of naturally occurring Ras effectors and acted as dominant-negative affinity reagents that block Ras signal transduction. Experiments with cancer cells showed that expression of these RBD variants inhibits Ras signaling, reducing cell growth and inducing apoptosis. Using these optimized RBD variants, we stratified patient-derived colorectal cancer organoids with known Ras mutational status according to their response to Ras inhibition. These results revealed that the presence of Ras mutations was insufficient to predict sensitivity to Ras inhibition, suggesting that not all of these tumors required Ras signaling for proliferation. In summary, by engineering the Ras/Raf interface of the CRAF-RBD, we identified potent and selective inhibitors of Ras in its active conformation that outcompete binding of Ras-signaling effectors.

PubMed: 32086379
DOI: 10.1074/jbc.RA119.011025

Experimental method

X-RAY DIFFRACTION (2.9 Å)