Andreas Boland

A DDX6-CNOT1 Complex and W-Binding Pockets in CNOT9 Reveal Direct Links between miRNA Target Recognition and Silencing

CCR4-NOT is a major effector complex in miRNA-mediated gene silencing. It is recruited to miRNA targets through interactions with tryptophan (W)-containing motifs in TNRC6/GW182 proteins and is required for both translational repression and degradation of miRNA targets. Here, we elucidate the structural basis for the repressive activity of CCR4-NOT and its interaction with TNRC6/GW182s. We show that the conserved CNOT9 subunit attaches to a domain of unknown function (DUF3819) in the CNOT1 scaffold. The resulting complex provides binding sites for TNRC6/GW182, and its crystal structure reveals tandem W-binding pockets located in CNOT9. We further show that the CNOT1 MIF4G domain interacts with the C-terminal RecA domain of DDX6, a translational repressor and decapping activator. The crystal structure of this complex demonstrates striking similarity to the eIF4G-eIF4A complex. Together, our data provide the missing physical links in a molecular pathway that connects miRNA target recognition with translational repression, deadenylation, and decapping.

Crystal Structure of the Mid-Piwi Lobe of a Eukaryotic Argonaute Protein

Argonaute proteins (AGOs) are essential effectors in RNA-mediated gene silencing pathways. They are characterized by a bilobal architecture, in which one lobe contains the N-terminal and PAZ domains and the other contains the MID and PIWI domains. Here, we present the first crystal structure of the MID-PIWI lobe from a eukaryotic AGO, the Neurospora crassa QDE-2 protein. Compared to prokaryotic AGOs, the domain orientation is conserved, indicating a conserved mode of nucleic acid binding. The PIWI domain shows an adaptable surface loop next to a eukaryote-specific α-helical insertion, which are both likely to contact the PAZ domain in a conformation-dependent manner to sense the functional state of the protein. The MID-PIWI interface is hydrophilic and buries residues that were previously thought to participate directly in the allosteric regulation of guide RNA binding. The interface includes the binding pocket for the guide RNA 5′ end, and residues from both domains contribute to binding. Accordingly, micro-RNA (miRNA) binding is particularly sensitive to alteration in the MID-PIWI interface in Drosophila melanogaster AGO1 in vivo. The structure of the QDE-2 MID-PIWI lobe provides molecular and mechanistic insight into eukaryotic AGOs and has significant implications for understanding the role of these proteins in silencing.

Crystal structure and ligand binding of the MID domain of a eukaryotic Argonaute protein

Argonaute (AGO) proteins are core components of RNA‐induced silencing complexes and have essential roles in RNA‐mediated gene silencing. They are characterized by a bilobal architecture, consisting of one lobe containing the amino‐terminal and PAZ domains and another containing the MID and PIWI domains. Except for the PAZ domain, structural information on eukaryotic AGO domains is not yet available. In this study, we report the crystal structure of the MID domain of the eukaryotic AGO protein QDE‐2 from Neurospora crassa. This domain adopts a Rossmann‐like fold and recognizes the 5′‐terminal nucleotide of a guide RNA in a manner similar to its prokaryotic counterparts. The 5′‐nucleotide‐binding site shares common residues with a second, adjacent ligand‐binding site, suggesting a mechanism for the cooperative binding of ligands to the MID domain of eukaryotic AGOs.

Fast native-SAD phasing for routine macromolecular structure determination

We describe a data collection method that uses a single crystal to solve X-ray structures by native SAD (single-wavelength anomalous diffraction). We solved the structures of 11 real-life examples, including a human membrane protein, a protein-DNA complex and a 266-kDa multiprotein-ligand complex, using this method. The data collection strategy is suitable for routine structure determination and can be implemented at most macromolecular crystallography synchrotron beamlines.

A direct interaction between DCP1 and XRN1 couples mRNA decapping to 5′ exonucleolytic degradation

The removal of the mRNA 5′ cap structure by the decapping enzyme DCP2 leads to rapid 5′→3′ mRNA degradation by XRN1, suggesting that the two processes are coordinated, but the coupling mechanism is unknown. DCP2 associates with the decapping activators EDC4 and DCP1. Here we show that XRN1 directly interacts with EDC4 and DCP1 in human and Drosophila melanogaster cells, respectively. In D. melanogaster cells, this interaction is mediated by the DCP1 EVH1 domain and a DCP1-binding motif (DBM) in the XRN1 C-terminal region. The NMR structure of the DCP1 EVH1 domain bound to the DBM reveals that the peptide docks at a conserved aromatic cleft, which is used by EVH1 domains to recognize proline-rich ligands. Our findings reveal a role for XRN1 in decapping and provide a molecular basis for the coupling of decapping to 5′→3′ mRNA degradation.

Structure of the PAN3 Pseudokinase Reveals the Basis for Interactions with the PAN2 Deadenylase and the GW182 Proteins

The PAN2-PAN3 deadenylase complex functions in general and miRNA-mediated mRNA degradation and is specifically recruited to miRNA targets by GW182/TNRC6 proteins. We describe the PAN3 adaptor protein crystal structure that, unexpectedly, forms intertwined and asymmetric homodimers. Dimerization is mediated by a coiled coil that links an N-terminal pseudokinase to a C-terminal knob domain. The PAN3 pseudokinase binds ATP, and this function is required for mRNA degradation in vivo. We further identified conserved surfaces required for mRNA degradation, including the binding surface for the PAN2 deadenylase on the knob domain. The most remarkable structural feature is the presence of a tryptophan-binding pocket at the dimer interface, which mediates binding to TNRC6C in human cells. Together, our data reveal the structural basis for the interaction of PAN3 with PAN2 and the recruitment of the PAN2-PAN3 complex to miRNA targets by TNRC6 proteins.

Structure and assembly of the NOT module of the human CCR4–NOT complex

The CCR4–NOT deadenylase complex is a master regulator of translation and mRNA stability. Its NOT module orchestrates recruitment of the catalytic subunits to target mRNAs. We report the crystal structure of the human NOT module formed by the CNOT1, CNOT2 and CNOT3 C-terminal (-C) regions. CNOT1-C provides a rigid scaffold consisting of two perpendicular stacks of HEAT-like repeats. CNOT2-C and CNOT3-C heterodimerize through their SH3-like NOT-box domains. The heterodimer is stabilized and tightly anchored to the surface of CNOT1 through an unexpected intertwined arrangement of peptide regions lacking defined secondary structure. These assembly peptides mold onto their respective binding surfaces and form extensive interfaces. Mutagenesis of individual interfaces and perturbation of endogenous protein ratios cause defects in complex assembly and mRNA decay. Our studies provide a structural framework for understanding the recruitment of the CCR4–NOT complex to mRNA targets.

Cryo-EM structure of a metazoan separase-securin complex at near-atomic resolution.

Separase is a caspase-family protease that initiates chromatid segregation by cleaving the kleisin subunits (Scc1 and Rec8) of cohesin, and regulates centrosome duplication and mitotic spindle function through cleavage of kendrin and Slk19. To understand the mechanisms of securin regulation of separase, we used single-particle cryo-electron microscopy (cryo-EM) to determine a near-atomic-resolution structure of the Caenorhabditis elegans separase–securin complex. Separase adopts a triangular-shaped bilobal architecture comprising an N-terminal tetratricopeptide repeat (TPR)-like α-solenoid domain docked onto the conserved C-terminal protease domain. Securin engages separase in an extended antiparallel conformation, interacting with both lobes. It inhibits separase by interacting with the catalytic site through a pseudosubstrate mechanism, thus revealing that in the inhibited separase–securin complex, the catalytic site adopts a conformation compatible with substrate binding. Securin is protected from cleavage because an aliphatic side chain at the P1 position represses protease activity by disrupting the organization of catalytic site residues.