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Revealing Functional Hotspots: Temperature-Dependent Crystallography of K-RAS Highlights Allosteric and Druggable
Samuel L Deck1,2, Megan Xu1,2, Shawn K Milano1,2
1Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853.
Abstract:
K-RAS mutations drive oncogenesis in multiple cancers, yet the lack of druggable sites has long hindered therapeutic development. Here, we use multi-temperature X-ray crystallography (MT-XRC) to capture functionally relevant K-RAS conformations across a temperature gradient, spanning cryogenic to physiological and even "fever" conditions, and show how cryogenic conditions may obscure key dynamic states as targets for new drug development. This approach revealed a temperature-dependent conformational landscape of K-RAS, shedding light on the dynamic nature of key regions. We identified significant conformational changes occurring at critical sites, including known allosteric and drug-binding pockets, which were hidden under cryogenic conditions but later discovered to be critically important for drug-protein interactions and inhibitor design. These structural changes align with regions previously highlighted by large-scale mutational studies as functionally significant. However, our MT-XRC analysis provides precise structural snapshots, capturing the exact conformations of these potentially important allosteric sites in unprecedented detail. Our findings underscore the necessity of advancing tools like MT-XRC to visualize conformational transitions that may be important in signal propagation which are missed by standard cryogenic XRC and to address hard-to-drug targets through rational drug design. This approach not only provides unique structural insights into K-RAS signaling events and identifies new potential sites to target with drug candidates but also establishes a powerful framework for discovering therapeutic opportunities against other challenging drug targets.
Insights
Multi-temperature X-ray crystallography reveals critical K-RAS protein dynamics missed at cryogenic temperatures. This breakthrough identifies new drug targets for hard-to-treat cancers by uncovering hidden allosteric sites.
Area of Science:
- Structural Biology
- Biophysics
- Oncology
Background:
- K-RAS mutations are key drivers in many cancers, but developing effective therapies has been challenging due to a lack of druggable targets.
- Standard X-ray crystallography often uses cryogenic temperatures, which may obscure functionally important protein dynamics.
Purpose of the Study:
- To investigate the temperature-dependent conformational landscape of K-RAS using multi-temperature X-ray crystallography (MT-XRC).
- To identify functionally relevant K-RAS conformations and allosteric sites that are obscured under cryogenic conditions.
Main Methods:
- Utilized multi-temperature X-ray crystallography (MT-XRC) to capture K-RAS structures across a range of temperatures, from cryogenic to physiological.
- Analyzed conformational changes in key regions, including known allosteric and drug-binding pockets.
Main Results:
- Revealed a temperature-dependent conformational landscape of K-RAS, highlighting dynamic states crucial for drug development.
- Identified significant conformational changes at critical sites, including allosteric pockets, which were hidden at cryogenic temperatures.
- Structural findings align with regions identified by large-scale mutational studies, providing precise snapshots of functionally significant allosteric sites.
Conclusions:
- MT-XRC is essential for visualizing conformational transitions missed by standard cryogenic X-ray crystallography, enabling the targeting of 'hard-to-drug' proteins.
- The study provides novel structural insights into K-RAS signaling and identifies new therapeutic targets for rational drug design.
- This framework can be applied to discover therapeutic opportunities against other challenging drug targets.
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