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Biophys J. 2019 Jan 22;116(2):179-183. doi: 10.1016/j.bpj.2018.12.011. Epub 2018 Dec 20.

Dynamics of Membrane-Bound G12V-KRAS from Simulations and Single-Molecule FRET in Native Nanodiscs.

Author information

1
Department of Integrative Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, Texas.
2
Department of Biochemistry and Molecular Biology, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, Texas; Biochemistry and Cell Biology Program, MD Anderson University of Texas Health Science Center at Houston, Graduate School of Biochemical Sciences, Houston, Texas.
3
Department of Biochemistry and Molecular Biology, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, Texas.
4
Department of Integrative Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, Texas; Biochemistry and Cell Biology Program, MD Anderson University of Texas Health Science Center at Houston, Graduate School of Biochemical Sciences, Houston, Texas.
5
Department of Integrative Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, Texas; Biochemistry and Cell Biology Program, MD Anderson University of Texas Health Science Center at Houston, Graduate School of Biochemical Sciences, Houston, Texas. Electronic address: alemayehu.g.abebe@uth.tmc.edu.

Abstract

Recent studies have shown that the small GTPase KRAS adopts multiple orientations with respect to the plane of anionic model membranes, whereby either the three C-terminal helices or the three N-terminal β-strands of the catalytic domain face the membrane. This has functional implications because, in the latter, the membrane occludes the effector-interacting surface. However, it remained unclear how membrane reorientation occurs and, critically, whether it occurs in the cell in which KRAS operates as a molecular switch in signaling pathways. Herein, using data from a 20 μs-long atomistic molecular dynamics simulation of the oncogenic G12V-KRAS mutant in a phosphatidylcholine/phosphatidylserine bilayer, we first show that internal conformational fluctuations of flexible regions in KRAS result in three distinct membrane orientations. We then show, using single-molecule fluorescence resonance energy transfer measurements in native lipid nanodiscs derived from baby hamster kidney cells, that G12V-KRAS samples three conformational states that correspond to the predicted orientations. The combined results suggest that relatively small energy barriers separate orientation states and that signaling-competent conformations dominate the overall population.

PMID:
30616834
PMCID:
PMC6350008
[Available on 2020-01-22]
DOI:
10.1016/j.bpj.2018.12.011

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