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J Biol Chem. 2017 Jul 28;292(30):12544-12559. doi: 10.1074/jbc.M117.785063. Epub 2017 Jun 16.

Flexible-body motions of calmodulin and the farnesylated hypervariable region yield a high-affinity interaction enabling K-Ras4B membrane extraction.

Author information

1
From the Cancer and Inflammation Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, NCI-Frederick, Frederick, Maryland 21702.
2
the Departments of Chemistry and.
3
the Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, and.
4
Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607, vadimg@uic.edu.
5
From the Cancer and Inflammation Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, NCI-Frederick, Frederick, Maryland 21702, NussinoR@helix.nih.gov.
6
the Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.

Abstract

In calmodulin (CaM)-rich environments, oncogenic KRAS plays a critical role in adenocarcinomas by promoting PI3K/Akt signaling. We previously proposed that at elevated calcium levels in cancer, CaM recruits PI3Kα to the membrane and extracts K-Ras4B from the membrane, organizing a K-Ras4B-CaM-PI3Kα ternary complex. CaM can thereby replace a missing receptor-tyrosine kinase signal to fully activate PI3Kα. Recent experimental data show that CaM selectively promotes K-Ras signaling but not of N-Ras or H-Ras. How CaM specifically targets K-Ras and how it extracts it from the membrane in KRAS-driven cancer is unclear. Obtaining detailed structural information for a CaM-K-Ras complex is still challenging. Here, using molecular dynamics simulations and fluorescence experiments, we observed that CaM preferentially binds unfolded K-Ras4B hypervariable regions (HVRs) and not α-helical HVRs. The interaction involved all three CaM domains including the central linker and both lobes. CaM specifically targeted the highly polybasic anchor region of the K-Ras4B HVR that stably wraps around CaM's acidic linker. The docking of the farnesyl group to the hydrophobic pockets located at both CaM lobes further enhanced CaM-HVR complex stability. Both CaM and K-Ras4B HVR are highly flexible molecules, suggesting that their interactions permit highly dynamic flexible-body motions. We, therefore, anticipate that the flexible-body interaction is required to extract K-Ras4B from the membrane, as conformational plasticity enables CaM to orient efficiently to the polybasic HVR anchor, which is partially diffused into the liquid-phase membrane. Our structural model of the CaM-K-Ras4B HVR association provides plausible clues to CaM's regulatory action in PI3Kα activation involving the ternary complex in cell proliferation signaling by oncogenic K-Ras.

KEYWORDS:

GTPase Kras (KRAS); KRAS-driven adenocarcinoma; PI3K; calmodulin (CaM); fluorescence; hypervariable region; molecular dynamics; nuclear magnetic resonance (NMR); post-translational modification (PTM); prenylation

PMID:
28623230
PMCID:
PMC5535030
DOI:
10.1074/jbc.M117.785063
[Indexed for MEDLINE]
Free PMC Article

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