Abstract
RAF activation requires coordinated interactions with both RAS and membrane lipids, yet the molecular basis of this process remains unclear. Using a bottom-up reconstitution approach, we show how coordinated protein–protein and protein–lipid interactions regulate membrane binding dynamics of RAF to drive its multistep activation. Within membrane environments, the RAS-binding domain (RBD) and cysteine-rich domain (CRD) exhibit cooperativity, with CRD-mediated phosphatidylserine binding stabilizing the RBD:RAS complex. Importantly, RAF remains membrane-bound through lateral rebinding to RAS, where a weak CRD–lipid interaction plays an essential role. The lateral rebinding extends RAF’s membrane dwell time under high RAS density conditions. This prolonged membrane residence may facilitate completion of RAF’s multistep activation. Given the high abundance of weak multivalent membrane interactions, lateral rebinding may be a common mechanism for regulating the activity of signaling proteins through sustained membrane retention.
Data availability
The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. The raw image data are available from the corresponding author upon request. These image data are not deposited in a public database because of their large file sizes. Source Data are provided with this paper. The structures of the CRAF/MEK1/14-3-3 complex in the autoinhibited conformation and the CRAF RBD-CRD/KRAS complex were obtained from the Protein Data Bank under accession codes 9MMP [https://doi.org/10.2210/pdb9mmp/pdb] and 6XI7 [https://doi.org/10.2210/pdb6xi7/pdb], respectively. Source data are provided with this paper.
Code availability
The custom MATLAB script used for TrackMate data processing, association kinetics, and step size distribution analysis is available on GitHub at https://github.com/youngkwanglee-mem/SPT_Kon_StepSize. The specific version of the code used for the analysis in this study has been deposited in Zenodo with the https://doi.org/10.5281/zenodo.1812651770.
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Acknowledgements
We thank Tom Huxford for his constructive feedback on the manuscript and helpful discussions on protein analysis. We acknowledge the Scientific Instrumentation Facilities in the Department of Chemistry and Biochemistry at the University of San Diego, and Scientific Instrument Specialist Joseph Avila, for providing access to the Jasco J-1100 Circular Dichroism Spectrophotometer (CD) and for technical support. This study was supported by an NSF CAREER Award MCB-2145852 to Y.K.L. A.J.S. was supported in part as a Fellow of the Rees-Stealy Research Foundation.
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A.J.S. expressed and purified all proteins, with assistance from K.T. and A.C. A.J.S., K.T., and Y.K.L. performed and analyzed ensemble and single-molecule binding assays, with assistance from J.G., A.C., and A.M. J.G. performed FCS experiments. Y.K.L. designed the experiments and directed the project. A.J.S. and Y.K.L. drafted the manuscript with input from all authors.
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Jimenez Salinas, A., Tevdorashvili, K., Grim, J. et al. Positive cooperativity between RAS-binding and cysteine-rich domains regulates RAF membrane binding kinetics via lateral rebinding. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69437-6
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DOI: https://doi.org/10.1038/s41467-026-69437-6
