Revealing KRas4b topology on the membrane surface

Shweta Shree, Mark A. McLean, Andrew G. Stephen, Stephen G. Sligar

Research output: Contribution to journalArticlepeer-review

Abstract

KRas4b is a membrane-bound regulatory protein belonging to the family of small GTPases that function as a molecular switch, facilitating signal transduction from activated membrane receptors to intracellular pathways controlling cell growth and proliferation. Oncogenic mutations locking KRas4b in the active GTP state are responsible for nearly 85% of all Ras-driven cancers. Understanding the membrane-bound state of KRas4b is crucial for designing new therapeutic approaches targeting oncogenic KRas-driven signaling pathways. Extensive research demonstrates the significant involvement of the membrane bilayer in Ras-effector interactions, with anionic lipids playing a critical role in determining protein conformations The preferred topology of KRas4b for interacting with signaling partners has been a long-time question. Computational studies suggest a membrane-proximal conformation, while other biophysical methods like neutron reflectivity propose a membrane-distal conformation. To address these gaps, we employed FRET measurements to investigate the conformation of KRas4b. Using fully post-translationally modified KRas4b, we designed a Nanodisc based FRET assay to study KRas4b-membrane interactions. We suggest an extended conformation of KRas4b relative to the membrane surface. Measurement of FRET donor - acceptor distances reveal that a negatively charged membrane surface weakly favors closer association with the membrane surface. Our findings provide insights into the role of anionic lipids in determining the dynamic conformations of KRas4b and shed light on the predominant conformation of its topology on lipid headgroups.

Original languageEnglish (US)
Pages (from-to)122-127
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume678
DOIs
StatePublished - Oct 20 2023

Keywords

  • Cancer signaling
  • KRas4b
  • Lipid specificity
  • Membrane topology
  • Nanodisc

ASJC Scopus subject areas

  • Molecular Biology
  • Biophysics
  • Biochemistry
  • Cell Biology

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