Poster abstracts
Poster number 14 submitted by Viraj Rana
Sampling the Wild-Type KRAS Excited State with AlphaFold3 via MSA Modulation
Viraj Rana (Chemistry ), Puyi Ma (Chemistry ), Sam Xing (Pharmacology ), Xiaolin Cheng, Rafael Bruschweiler (Chemistry )
Abstract:
Protein excited states are transient conformations that occupy local minima on the energy landscape and are typically populated on the μs-ms timescale at low abundance1. Despite their functional relevance in catalysis, signaling, and allostery, their structural characterization remains challenging due to their short lifetimes and limited population1,2. KRAS is a small GTPase that regulates cell growth and differentiation and is frequently mutated in cancer. With mutations driving ~25% of cancers, KRAS has remained difficult to target therapeutically, in part due to its dynamic conformational landscape, which has limited structural characterization of its excited state. Here, we address this gap by identifying candidate KRAS excited-state conformations. These excited state conformations are in GTP-bound form, where KRAS predominantly adopts an active state (~90%) but also samples a low-populated excited state (~10%) that exhibits structural features similar to the GDP-bound form, particularly in the Switch I (residues 30-38) and Switch II (residues 60-76) regions3. We explore whether AlphaFold3 can sample candidate conformations consistent with the KRAS excited state by modifying the multiple sequence alignment (MSA)4. We find that specific MSA perturbations enable AlphaFold3 to generate structural ensembles spanning active-like, inactive-like, and intermediate conformations, including candidates consistent with an excited-state-like geometry in the switch regions. Molecular dynamics simulations will be used as a next step to evaluate the stability and conformational sampling of these models. This combined approach provides a framework for probing low-populated KRAS states computationally and for generating testable structural hypotheses that can be compared against experimental observables. More broadly, it evaluates the extent to which AlphaFold3, when guided via MSA modulation, can access functionally relevant regions of the KRAS conformational landscape.
References:
1. Baldwin, A. J. & Kay, L. E. NMR spectroscopy brings invisible protein states into focus. Nat. Chem. Biol. 5, 808–814 (2009).
2. Sekhar, A. & Kay, L. E. NMR paves the way for atomic level descriptions of sparsely populated, transiently formed biomolecular conformers. Proc. Natl. Acad. Sci. U. S. A. 110, 12867–12874 (2013).
3. Hansen, A. L., Xiang, X., Yuan, C., Bruschweiler-Li, L. & Brüschweiler, R. Excited-state observation of active K-Ras reveals differential structural dynamics of wild-type versus oncogenic G12D and G12C mutants. Nat. Struct. Mol. Biol. 30, 1446–1455 (2023).
4. Abramson, J. et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 630, 493–500 (2024).
Keywords: KRAS, AlphaFold3, Multiple Sequence Alignment