K-Ras G-domain binding with signaling lipid phosphatidylinositol (4,5)-phosphate (PIP2): membrane association, protein orientation, and function

J Biol Chem. 2019 Apr 26;294(17):7068-7084. doi: 10.1074/jbc.RA118.004021. Epub 2019 Feb 21.


Ras genes potently drive human cancers, with mutated proto-oncogene GTPase KRAS4B (K-Ras4B) being the most abundant isoform. Targeted inhibition of oncogenic gene products is considered the "holy grail" of present-day cancer therapy, and recent discoveries of small-molecule KRas4B inhibitors were made thanks to a deeper understanding of the structure and dynamics of this GTPase. Because interactions with biological membranes are key for Ras function, Ras-lipid interactions have become a major focus, especially because such interactions evidently involve both the Ras C terminus for lipid anchoring and its G-protein domain. Here, using NMR spectroscopy and molecular dynamics simulations complemented by biophysical- and cell-biology assays, we investigated the interaction between K-Ras4B with the signaling lipid phosphatidylinositol (4,5)-phosphate (PIP2). We discovered that the β2 and β3 strands as well as helices 4 and 5 of the GTPase G-domain bind to PIP2 and identified the specific residues in these structural elements employed in these interactions, likely occurring in two K-Ras4B orientation states relative to the membrane. Importantly, we found that some of these residues known to be oncogenic when mutated (D47K, D92N, K104M, and D126N) are critical for K-Ras-mediated transformation of fibroblast cells, but do not substantially affect basal and assisted nucleotide hydrolysis and exchange. Moreover, the K104M substitution abolished localization of K-Ras to the plasma membrane. The findings suggest that specific G-domain residues can critically regulate Ras function by mediating interactions with membrane-associated PIP2 lipids; these insights that may inform the future design of therapeutic reagents targeting Ras activity.

Keywords: PIP2; Ras GTPase; Ras protein; cancer; cell signaling; cell transformation; molecular dynamics; nuclear magnetic resonance (NMR); phosphoinositide; protein structure; protein-membrane interactions.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Humans
  • Membrane Lipids / metabolism*
  • Nuclear Magnetic Resonance, Biomolecular
  • Phosphoinositide Phospholipase C / metabolism*
  • Protein Binding
  • Protein Conformation
  • Protein Domains
  • Proto-Oncogene Mas
  • Proto-Oncogene Proteins p21(ras) / chemistry
  • Proto-Oncogene Proteins p21(ras) / metabolism*


  • MAS1 protein, human
  • Membrane Lipids
  • Proto-Oncogene Mas
  • Phosphoinositide Phospholipase C
  • Proto-Oncogene Proteins p21(ras)

Associated data

  • PDB/4TQ9
  • PDB/4DSO