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The Structural Basis of Oncogenic Mutations G12, G13 and Q61 in Small GTPase K-Ras4B.

ABSTRACT: Ras mediates cell proliferation, survival and differentiation. Mutations in K-Ras4B are predominant at residues G12, G13 and Q61. Even though all impair GAP-assisted GTP ? GDP hydrolysis, the mutation frequencies of K-Ras4B in human cancers vary. Here we aim to figure out their mechanisms and differential oncogenicity. In total, we performed 6.4 ?s molecular dynamics simulations on the wild-type K-Ras4B (K-Ras4B(WT)-GTP/GDP) catalytic domain, the K-Ras4B(WT)-GTP-GAP complex, and the mutants (K-Ras4B(G12C/G12D/G12V)-GTP/GDP, K-Ras4B(G13D)-GTP/GDP, K-Ras4B(Q61H)-GTP/GDP) and their complexes with GAP. In addition, we simulated 'exchanged' nucleotide states. These comprehensive simulations reveal that in solution K-Ras4B(WT)-GTP exists in two, active and inactive, conformations. Oncogenic mutations differentially elicit an inactive-to-active conformational transition in K-Ras4B-GTP; in K-Ras4B(G12C/G12D)-GDP they expose the bound nucleotide which facilitates the GDP-to-GTP exchange. These mechanisms may help elucidate the differential mutational statistics in K-Ras4B-driven cancers. Exchanged nucleotide simulations reveal that the conformational transition is more accessible in the GTP-to-GDP than in the GDP-to-GTP exchange. Importantly, GAP not only donates its R789 arginine finger, but stabilizes the catalytically-competent conformation and pre-organizes catalytic residue Q61; mutations disturb the R789/Q61 organization, impairing GAP-mediated GTP hydrolysis. Together, our simulations help provide a mechanistic explanation of key mutational events in one of the most oncogenic proteins in cancer.

SUBMITTER: Lu S

PROVIDER: S-EPMC4763299 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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The Structural Basis of Oncogenic Mutations G12, G13 and Q61 in Small GTPase K-Ras4B.

Lu Shaoyong S Jang Hyunbum H Nussinov Ruth R Zhang Jian J

Scientific reports 20160223

Ras mediates cell proliferation, survival and differentiation. Mutations in K-Ras4B are predominant at residues G12, G13 and Q61. Even though all impair GAP-assisted GTP → GDP hydrolysis, the mutation frequencies of K-Ras4B in human cancers vary. Here we aim to figure out their mechanisms and differential oncogenicity. In total, we performed 6.4 μs molecular dynamics simulations on the wild-type K-Ras4B (K-Ras4B(WT)-GTP/GDP) catalytic domain, the K-Ras4B(WT)-GTP-GAP complex, and the mutants (K-R ...[more]

PMID: 26902995

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Project description:Ras recruits and activates effectors that transmit receptor-initiated signals. Monomeric Ras can bind Raf; however, Raf's activation requires dimerization, which can be facilitated by Ras dimerization. Previously, we showed that active K-Ras4B dimerizes in silico and in vitro through two major interfaces: (i) β-interface, mapped to Switch I and effector-binding regions, (ii) α-interface at the allosteric lobe. Here, we chose constitutively active K-Ras4B as our control and two double mutants (K101D and R102E; and R41E and K42D) in the α- and β-interfaces. Two of the mutations are from The Cancer Genome Atlas (TCGA) and the Catalogue Of Somatic Mutations In Cancer (COSMIC) data sets. R41 and R102 are found in several adenocarcinomas in Ras isoforms. We performed site-directed mutagenesis, cellular localization experiments, and molecular dynamics (MD) simulations to assess the impact of the mutations on K-Ras4B dimerization and function. α-interface K101D/R102E double mutations reduced dimerization but only slightly reduced downstream phosphorylated extracellular signal-regulated kinase (ERK) (pERK) levels. While β-interface R41E/K42D double mutations did not interfere with dimerization, they almost completely blocked K-Ras4B-mediated ERK phosphorylation. Both double mutations increased downstream phosphorylated Akt (pAkt) levels in cells. Changes in pERK and pAkt levels altered ERK- and Akt-regulated gene expressions, such as EGR1, JUN, and BCL2L11. These results underscore the role of the α-interface in K-Ras4B homodimerization and the β-surface in effector binding. MD simulations highlight that the membrane and hypervariable region (HVR) interact with both α- and β-interfaces of K-Ras4B mutants, respectively, inhibiting homodimerization and probably effector binding. Mutations at both interfaces interfered with mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase signaling but in different forms and extents. We conclude that dimerization is not necessary but enhances downstream MAPK signaling.

Dataset Information

The Structural Basis of Oncogenic Mutations G12, G13 and Q61 in Small GTPase K-Ras4B.

Publications

The Structural Basis of Oncogenic Mutations G12, G13 and Q61 in Small GTPase K-Ras4B.

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