Structure of a SMG8–SMG9 complex identifies a G-domain heterodimer in the NMD effector proteins

Abstract

Nonsense-mediated mRNA decay (NMD) is a eukaryotic mRNA degradation pathway involved in surveillance and posttranscriptional regulation, and executed by the concerted action of several trans-acting factors. The SMG1 kinase is an essential NMD factor in metazoans and is associated with two recently identified and yet poorly characterized proteins, SMG8 and SMG9. We determined the 2.5 Å resolution crystal structure of a SMG8-SMG9 core complex from C. elegans. We found that SMG8-SMG9 is a G-domain heterodimer with architectural similarities to the dynamin-like family of GTPases such as Atlastin and GBP1. The SMG8-SMG9 heterodimer forms in the absence of nucleotides, with interactions conserved from worms to humans. Nucleotide binding occurs at the G domain of SMG9 but not of SMG8. Fitting the GDP-bound SMG8-SMG9 structure in EM densities of the human SMG1-SMG8-SMG9 complex raises the possibility that the nucleotide site of SMG9 faces SMG1 and could impact the kinase conformation and/or regulation.

Cryo-EM structure of SMG1–SMG8–SMG9 complex

“…SMG8-SMG9 heterodimer stably associates with SMG1 and the C-terminal region of SMG8 inhibits SMG1 kinase activity through covering the catalytic pocket. By comparing with the previously reported structure, 28 we proposed a mechanism for activation of SMG1 kinase upon GTP hydrolysis of the SMG9 GTPase. The structural and biochemical analyses together provide structural insights into the assembly of SMG1C complex and the regulation of SMG1 kinase activity.…”

“…The structural observation is consistent with the previous study that double mutations M390R/Y515R of human SMG9 impaired the interaction with SMG8. 28 Human SMG9 has undetectable GTPase activity in the context of SMG1C complex because GTP is stably maintained during protein purification ( Fig. 4a), suggesting that SMG1C adopts a GTP-bound state.…”

“…S2, 3, Table S1 and Movies S1, 2). The structural models were built by homology modeling and manually building into the EM density using the structures of mTOR 30 and C. elegans SMG8-SMG9 complex 28 as templates.…”

“…Crystal structure of C. elegans SMG8-SMG9 core complex reveals that SMG8-SMG9 heterodimer is formed through the contacts between a C-terminal guanine nucleotidebinding (G) domain of SMG9 and an N-terminal G-like domain of SMG8. 28 Previous electron microscopic studies revealed lowresolution density map of SMG1 and SMG1C. 25,27,29 However, the mechanisms for complex assembly and regulation of SMG1 kinase by SMG8 and SMG9 remain largely unknown.…”

Nonsense-Mediated mRNA Decay: Pathologies and the Potential for Novel Therapeutics

“…Three-dimensional protein structures can reveal the precise interactions defining the protein-protein interface for in silico drug design, which can be targeted for drug discovery [109][110][111]. These protein-protein structures available in the Protein Data Bank (http://www.rcsb.org/pdb) [111] for NMD pathway are ( Figure 2 and Table 1): UPF1-UPF2 [34], UPF2-UPF3b [36], SMG5-SMG7 [103,105,112], SMG8-SMG9 [104,106], SMG1-SMG8-SMG9 [107] and the EJC (Mago-Y14-eIF4AIII-Barentsz-UPF3b) [108]. Figure 2, illustrates different protein-protein or protein-RNA interactions from the NMD pathways that could represent a base or the platform to design inhibitors or peptide-like molecules.…”