9zr7
Cryo-EM structure of NRAS(Q61K)-BRIL fusion in complex with Fab(BAG2) and Monobody(Mb24)
Structural highlights
FunctionRASN_CAVPO Signal transducer in the Ras-MAPK signaling pathway that regulates cell proliferation and survival (By similarity). Ras proteins bind GDP/GTP and possess intrinsic GTPase activity (By similarity). Recognized by LZTR1 that mediates its ubiquitination by a BCR (BTB-CUL3-RBX1) E3 ubiquitin-protein ligase complex (By similarity).[UniProtKB:P01111]C562_ECOLX Electron-transport protein of unknown function. Publication Abstract from PubMedSmall GTPases play important roles in cellular signaling. Due to their small sizes ( approximately 21 kDa), structural studies of small GTPases have been predominantly performed using x-ray crystallography in which crystal lattice contacts made it challenging to define unperturbed conformations of the key switch regions. Here, we developed a protein-engineering strategy that enables cryo-EM analysis of small soluble proteins and applied to RAS. We fused the C-terminal alpha5 helix of the RAS globular domain to a small protein BRIL by forming a continuous helix, which leaves most RAS surfaces exposed to the solvent and unperturbed, followed by the complex formation with an anti-BRIL Fab. This engineered complex with an increased molecular weight, termed "RAS-lollipop", enabled single-particle cryo-EM of RAS. Using this approach, we determined the cryo-EM structure of NRAS, whose structural studies using crystallography have been the least successful among the RAS isoforms. We revealed the conformations of the switch region and alpha 5 helix that differ from those observed in published crystal structures, and also defined the binding site of an NRAS-specific monobody. We uncovered an unexpected surfactant-like property of this monobody, which reduces orientation biases of particles on cryo-EM grids. Together, this work establishes a platform for visualizing small GTPases and potentially other small proteins with minimal perturbation of their surfaces. Protein Engineering-Enabled Cryo-EM Investigation of Small GTPases.,Hu Z, Patel UR, Glasser E, Koide A, Koide S J Mol Biol. 2026 Sep 1;438(17):169860. doi: 10.1016/j.jmb.2026.169860. Epub 2026 , May 13. PMID:42134495[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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