Spatiotemporal Analysis of K‑Ras Plasma Membrane Interactions Reveals Multiple High Order Homo-oligomeric Complexes

Self-assembly of plasma membrane-associated Ras GTPases has major implications to the regulation of cell signaling. However, the structural basis of homo-oligomerization and the fractional distribution of oligomeric states remained undetermined. We have addressed these issues by deciphering the dist...

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Bibliographic Details
Published in:Journal of the American Chemical Society 2017-09, Vol.139 (38), p.13466-13475
Main Authors: Sarkar-Banerjee, Suparna, Sayyed-Ahmad, Abdallah, Prakash, Priyanka, Cho, Kwang-Jin, Waxham, M. Neal, Hancock, John F, Gorfe, Alemayehu A
Format: Article
Language:English
Subjects:
Animals
Cell Membrane - metabolism
Hominidae
Humans
Models, Molecular
Mutant Proteins - chemistry
Mutant Proteins - genetics
Mutant Proteins - metabolism
Mutant Proteins - ultrastructure
Protein Multimerization
ras Proteins - chemistry
ras Proteins - genetics
ras Proteins - metabolism
ras Proteins - ultrastructure
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Summary:Self-assembly of plasma membrane-associated Ras GTPases has major implications to the regulation of cell signaling. However, the structural basis of homo-oligomerization and the fractional distribution of oligomeric states remained undetermined. We have addressed these issues by deciphering the distribution of dimers and higher-order oligomers of K-Ras4B, the most frequently mutated Ras isoform in human cancers. We focused on the constitutively active G12V K-Ras and two of its variants, K101E and K101C/E107C, which respectively destabilize and stabilize oligomers. Using raster image correlation spectroscopy and number and brightness analysis combined with fluorescence recovery after photobleaching, fluorescence correlation spectroscopy and electron microscopy in live cells, we show that G12V K-Ras exists as a mixture of monomers, dimers and larger oligomers, while the K101E mutant is predominantly monomeric and K101C/E107C is dominated by oligomers. This observation demonstrates the ability of K-Ras to exist in multiple oligomeric states whose population can be altered by interfacial mutations. Using molecular modeling and simulations we further show that K-Ras uses two partially overlapping interfaces to form compositionally and topologically diverse oligomers. Our results thus provide the first detailed insight into the multiplicity, structure, and membrane organization of K-Ras homomers.
ISSN:0002-7863
1520-5126
DOI:10.1021/jacs.7b06292