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Updated: May 27, 2025
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Published on: July 25, 2013
De novo design of Ras isoform selective binders
Jason Z Zhang1,2,3, Xinting Li1,2, Alexa Rane Batingana4
1Department of Biochemistry, University of Washington, Seattle, Washington 98195, United States.
Abstract:
The proto-oncogene Ras which governs diverse intracellular pathways has four major isoforms (KRAS4A, KRAS4B, HRAS, and NRAS) with substantial sequence homology and similar in vitro biochemistry. There is considerable interest in investigating the roles of these independently as their association with different cancers vary, but there are few Ras isoform-specific binding reagents as the only significant sequence differences are in their disordered and highly charged C-termini which have been difficult to elicit antibodies against. To overcome this limitation, we use deep learning-based methods to de novo design Ras isoform-specific binders (RIBs) for all major Ras isoforms that specifically target the Ras C-terminus. The RIBs bind to their target Ras isoforms both in vitro and in cells with remarkable specificity, disrupting their membrane localization and inhibiting Ras activity, and should contribute to dissecting the distinct roles of Ras isoforms in biology and disease.
Insights
Researchers developed novel Ras isoform-specific binders (RIBs) using deep learning. These binders target the Ras C-terminus, enabling the study of distinct Ras isoform roles in cancer and cellular pathways.
Area of Science:
- Molecular Biology
- Oncology
- Bioinformatics
- Protein Engineering
Background:
- The proto-oncogene Ras regulates critical intracellular pathways, with four major isoforms (KRAS4A, KRAS4B, HRAS, and NRAS) exhibiting high sequence homology.
- Investigating individual Ras isoform functions is crucial due to their varied associations with different cancers.
- A significant limitation in Ras research is the scarcity of isoform-specific binding reagents, primarily due to challenges in targeting their divergent C-termini.
Purpose of the Study:
- To overcome the lack of isoform-specific reagents for Ras proteins.
- To design and validate novel binding molecules that specifically target the C-termini of Ras isoforms.
- To enable the dissection of distinct biological and pathological roles of individual Ras isoforms.
Main Methods:
- Utilized deep learning-based methods for *de novo* design of protein binders.
- Engineered Ras isoform-specific binders (RIBs) targeting the unique C-terminal regions of KRAS4A, KRAS4B, HRAS, and NRAS.
- Validated the specificity and functionality of designed RIBs *in vitro* and in cellular models.
Main Results:
- Successfully designed and generated Ras isoform-specific binders (RIBs) targeting the Ras C-terminus.
- Demonstrated high specificity of RIBs for their cognate Ras isoforms both *in vitro* and within cells.
- Observed that RIBs disrupt Ras membrane localization and inhibit Ras activity, confirming their functional impact.
Conclusions:
- Deep learning enables the *de novo* design of highly specific protein binders for challenging targets like Ras C-termini.
- The developed RIBs are valuable tools for distinguishing the functions of Ras isoforms in biological processes.
- These RIBs hold potential for advancing research into Ras-driven diseases, including various cancers.
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