Samuel L Deck, Megan Xu, Shawn K Milano, Richard A Cerione
{"title":"Revealing Functional Hotspots: Temperature-Dependent Crystallography of K-RAS Highlights Allosteric and Druggable Sites.","authors":"Samuel L Deck, Megan Xu, Shawn K Milano, Richard A Cerione","doi":"10.1101/2025.02.27.639303","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2025-02-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11888411/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"bioRxiv : the preprint server for biology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1101/2025.02.27.639303","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
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.