PDBsum entry 6ntc

Transferase/protein binding

PDB id: 6ntc

Contents

Protein chains

168 a.a.

63 a.a.

Ligands

GNP

GOL

Metals

_MG

Waters ×5

PDB id:

6ntc

Name:

Transferase/protein binding

Title:

Crystal structure of g12v hras-gppnhp bound in complex with the engineered rbd variant 1 of craf kinase protein

Structure:

Gtpase hras. Chain: a. Synonym: h-ras-1,ha-ras,transforming protein p21,c-h-ras,p21ras. Engineered: yes. Mutation: yes. Raf proto-oncogene serine/threonine-protein kinase. Chain: b. Synonym: proto-oncogenE C-raf,craf,raf-1. Engineered: yes.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: hras, hras1. Expressed in: escherichia coli. Expression_system_taxid: 562. Gene: raf1, raf. Expression_system_taxid: 562

Resolution:

2.90Å R-factor: 0.253 R-free: 0.278

Authors:

P.Maisonneuve,I.Kurinov,S.Wiechmann,A.Ernst,F.Sicheri

Key ref:

S.Wiechmann et al. (2020). Conformation-specific inhibitors of activated Ras GTPases reveal limited Ras dependency of patient-derived cancer organoids. J Biol Chem, 295, 4526-4540. PubMed id: 32086379 DOI: 10.1074/jbc.RA119.011025

Date:

28-Jan-19 Release date: 04-Mar-20

Protein chain

P01112  (RASH_HUMAN) -  GTPase HRas from Homo sapiens

Seq: 189 a.a.

Struc: 168 a.a.*

Protein chain

P04049  (RAF1_HUMAN) -  RAF proto-oncogene serine/threonine-protein kinase from Homo sapiens

Seq: 648 a.a.

Struc: 63 a.a.*

* PDB and UniProt seqs differ at 11 residue positions (black crosses)

Enzyme reactions

• Enzyme class 2: Chain A: E.C.3.6.5.2 - small monomeric GTPase.
• Reaction: GTP + H2O = GDP + phosphate + H⁺

GTP + H2O = GDP (Bound ligand (Het Group name = GNP) matches with 81.82% similarity) + phosphate + H(+)

• Enzyme class 3: Chain B: E.C.2.7.11.1 - non-specific serine/threonine protein kinase.
• Reaction:
  1. L-seryl-[protein] + ATP = O-phospho-L-seryl-[protein] + ADP + H⁺
  2. L-threonyl-[protein] + ATP = O-phospho-L-threonyl-[protein] + ADP + H⁺

L-seryl-[protein] + ATP = O-phospho-L-seryl-[protein] (Bound ligand (Het Group name = GNP) matches with 78.79% similarity) + ADP + H(+)

L-threonyl-[protein] + ATP = O-phospho-L-threonyl-[protein] (Bound ligand (Het Group name = GNP) matches with 78.79% similarity) + ADP + H(+)

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1074/jbc.RA119.011025

J Biol Chem 295:4526-4540 (2020)

PubMed id: 32086379

Conformation-specific inhibitors of activated Ras GTPases reveal limited Ras dependency of patient-derived cancer organoids.

S.Wiechmann, P.Maisonneuve, B.M.Grebbin, M.Hoffmeister, M.Kaulich, H.Clevers, K.Rajalingam, I.Kurinov, H.F.Farin, F.Sicheri, A.Ernst.

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.