1. Cryo-EM reconstructions of inhibitor-bound SMG1 kinase reveal an autoinhibitory state dependent on SMG8
- Author
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Langer, Lukas M, Bonneau, Fabien, Gat, Yair, and Conti, Elena
- Subjects
QH301-705.5 ,Cryo-electron microscopy ,Structural Biology and Molecular Biophysics ,Science ,Nonsense-mediated decay ,nonsense-mediated mRNA decay ,Protein Serine-Threonine Kinases ,General Biochemistry, Genetics and Molecular Biology ,mRNA surveillance ,PIKK ,Humans ,Phosphorylation ,Biology (General) ,Kinase activity ,General Immunology and Microbiology ,biology ,Kinase ,Chemistry ,General Neuroscience ,Cryoelectron Microscopy ,Intracellular Signaling Peptides and Proteins ,Active site ,General Medicine ,RNA Helicase A ,Small molecule ,Cell biology ,HEK293 Cells ,Structural biology ,Trans-Activators ,biology.protein ,Medicine ,Research Advance ,RNA Helicases ,Function (biology) ,Human - Abstract
The PI3K-related kinase (PIKK) SMG1 monitors the progression of metazoan nonsense-mediated mRNA decay (NMD) by phosphorylating the RNA helicase UPF1. Previous work has shown that the activity of SMG1 is impaired by small molecule inhibitors, is reduced by the SMG1 interactors SMG8 and SMG9, and is downregulated by the so-called SMG1 insertion domain. However, the molecular basis for this complex regulatory network has remained elusive. Here, we present cryo-electron microscopy reconstructions of human SMG1-9 and SMG1-8-9 complexes bound to either a SMG1 inhibitor or a non-hydrolyzable ATP analog at overall resolutions ranging from 2.8 to 3.6 angstrom. These structures reveal the basis with which a small molecule inhibitor preferentially targets SMG1 over other PIKKs. By comparison with our previously reported substrate-bound structure (Langer et al.,2020), we show that the SMG1 insertion domain can exert an autoinhibitory function by directly blocking the substrate-binding path as well as overall access to the SMG1 kinase active site. Together with biochemical analysis, our data indicate that SMG1 autoinhibition is stabilized by the presence of SMG8. Our results explain the specific inhibition of SMG1 by an ATP-competitive small molecule, provide insights into regulation of its kinase activity within the NMD pathway, and expand the understanding of PIKK regulatory mechanisms in general. Acknowledgements: Daniel Bollschweiler and Tillman Schäfer at the MPIB cryo-EM facility for help with EM data collection and Barbara Steigenberger and Elisabeth Weyher at MPIB biochemistry core facility for carrying out mass spectrometry.
- Published
- 2021