Structural insights into Drosophila-C3PO complex assembly and 'Dynamic Side Port' model in substrate entry and release.
In Drosophila and human, component 3 promoter of RISC (C3PO), a heteromeric complex, enhances RISC assembly and promotes RISC activity. Here, we report crystal structure of full-length Drosophila C3PO (E126Q), an inactive C3PO mutant displaying much weaker RNA binding ability, at 2.1 A resolution. In addition, we also report the ... cryo-EM structures of full-length Drosophila C3PO (E126Q), C3PO (WT) and SUMO-C3PO (WT, sumo-TRAX + Translin) particles trapped at different conformations at 12, 19.7 and 12.8 A resolutions, respectively. Crystal structure of C3PO (E126Q) displays a half-barrel architecture consisting of two Trax/Translin heterodimers, whereas cryo-EM structures of C3PO (E126Q), C3PO (WT) and SUMO-C3PO (WT) adopt a closed football-like shape with a hollow interior cavity. Remarkably, both cryo-EM structures of Drosophila C3PO (E126Q) and Drosophila SUMO-C3PO (WT) particles contain a wide side port (?25 A × ?30 A versus ?15 A × ?20 A) for RNA substrate entry and release, formed by a pair of anti-parallel packed long ?1 helices of TRAX subunits. Notably, cryo-EM structure of SUMO-C3PO showed that four copies of extra densities belonging to N-terminal SUMO tag are located at the outside shell of SUMO-C3PO particle, which demonstrated that the stoichiometry of TRAX/Translin for the in vitro expressed and assembled full-length Drosophila-SUMO-C3PO particle is 4:4, suggesting Drosophila C3PO is composed by TRAX/translin at a ratio of 4:4. Remarkably, the comparison of the cryo-EM structures suggests that the C3PO side ports regulated by ?1 helices of TRAX molecules are highly dynamic. Hence, we propose that C3PO particles could adopt a 'Dynamic Side Port' model to capture/digest nucleic acid duplex substrate and release the digested fragments through the dynamic side ports.
Mesh Terms:
Animals, Carrier Proteins, Cryoelectron Microscopy, Crystallography, X-Ray, DNA-Binding Proteins, Drosophila Proteins, Drosophila melanogaster, Humans, Models, Molecular, Multiprotein Complexes, Mutation, Nucleic Acid Conformation, Particle Size, Protein Binding, Protein Conformation, RNA, RNA-Induced Silencing Complex, Sumoylation
Animals, Carrier Proteins, Cryoelectron Microscopy, Crystallography, X-Ray, DNA-Binding Proteins, Drosophila Proteins, Drosophila melanogaster, Humans, Models, Molecular, Multiprotein Complexes, Mutation, Nucleic Acid Conformation, Particle Size, Protein Binding, Protein Conformation, RNA, RNA-Induced Silencing Complex, Sumoylation
Nucleic Acids Res
Date: Dec. 19, 2017
PubMed ID: 29860349
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