Related Experiment Video
Updated: Feb 16, 2026
Electroporation of Functional Bacterial Effectors into Mammalian Cells
Published on: January 19, 2015
Exceptionally high-affinity Ras binders that remodel its effector domain
John H McGee1,2,3, So Youn Shim2,3,4, Seung-Joo Lee2
1From the Departments of Molecular and Cellular Biology.
Abstract:
The Ras proteins are aberrantly activated in a wide range of human cancers, often endowing tumors with aggressive properties and resistance to therapy. Decades of effort to develop direct Ras inhibitors for clinical use have thus far failed, largely because of a lack of adequate small-molecule-binding pockets on the Ras surface. Here, we report the discovery of Ras-binding miniproteins from a naïve library and their evolution to afford versions with midpicomolar affinity to Ras. A series of biochemical experiments indicated that these miniproteins bind to the Ras effector domain as dimers, and high-resolution crystal structures revealed that these miniprotein dimers bind Ras in an unprecedented mode in which the Ras effector domain is remodeled to expose an extended pocket that connects two isolated pockets previously found to engage small-molecule ligands. We also report a Ras point mutant that stabilizes the protein in the open conformation trapped by these miniproteins. These findings provide new tools for studying Ras structure and function and present opportunities for the development of both miniprotein and small-molecule inhibitors that directly target the Ras proteins.
Insights
Researchers discovered novel miniproteins that bind to Ras proteins, a key factor in many cancers. These miniproteins bind Ras in a unique way, opening new avenues for developing cancer therapies.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Ras proteins are frequently activated in human cancers, contributing to tumor aggressiveness and therapeutic resistance.
- Direct inhibition of Ras proteins has been challenging due to the lack of suitable binding pockets on their surface.
Purpose of the Study:
- To discover and develop novel Ras-binding molecules.
- To investigate the binding mechanism of these molecules to Ras.
- To explore new therapeutic strategies targeting Ras proteins.
Main Methods:
- Screening of a naïve library to identify Ras-binding miniproteins.
- Protein engineering to enhance miniprotein affinity for Ras.
- Biochemical assays to characterize miniprotein-Ras interactions.
- High-resolution crystal structures to determine the binding mode.
Main Results:
- Discovery of miniproteins with midpicomolar affinity for Ras.
- Demonstration that miniproteins bind Ras as dimers, inducing an unprecedented conformational change.
- Identification of a Ras point mutant that stabilizes the open conformation bound by miniproteins.
Conclusions:
- Miniproteins offer a new class of binders for Ras proteins.
- The unique binding mode reveals a druggable extended pocket on the Ras effector domain.
- These findings provide tools for Ras research and enable development of novel Ras inhibitors.
Related Concept Videos
02:55Exceptions to the Octet Rule
03:07Electron Affinity
02:38The Ras Gene
Ras is a...
02:54Nucleosome Remodeling
Nucleosome remodeling complex
Eukaryotic cells have specialized enzymes called ATP-dependent nucleosome remodeling enzymes. These enzymes...
01:41Affinity and Avidity
01:24Small GTPases - Ras and Rho
Three regulatory proteins control their activity: