Methylation of adenosines at the N6 position (m6A) is a dynamic and abundant epitranscriptomic mark that regulates critical aspects of eukaryotic RNA metabolism in numerous biological processes. The RNA methyltransferases METTL3 and METTL14 are components of a multisubunit m6A writer complex whose enzymatic activity is substantially higher than the activities of METTL3 or METTL14 alone. The molecular mechanism underpinning this synergistic effect is poorly understood. Here we report the crystal structure of the catalytic core of the human m6A writer complex comprising METTL3 and METTL14. The structure reveals the heterodimeric architecture of the complex and donor substrate binding by METTL3. Structure-guided mutagenesis indicates that METTL3 is the catalytic subunit of the complex, whereas METTL14 has a degenerate active site and plays non-catalytic roles in maintaining complex integrity and substrate RNA binding. These studies illuminate the molecular mechanism and evolutionary history of eukaryotic m6A modification in post-transcriptional genome regulation.DOI: http://dx.doi.org/10.7554/eLife.18434.001

The 26S proteasome is a 2.5 MDa molecular machine that executes the degradation of substrates of the ubiquitin-proteasome pathway. The molecular architecture of the 26S proteasome was recently established by cryo-EM approaches. For a detailed understanding of the sequence of events from the initial binding of polyubiquitylated substrates to the translocation into the proteolytic core complex, it is necessary to move beyond static structures and characterize the conformational landscape of the 26S proteasome. To this end we have subjected a large cryo-EM dataset acquired in the presence of ATP and ATP-γS to a deep classification procedure, which deconvolutes coexisting conformational states. Highly variable regions, such as the density assigned to the largest subunit, Rpn1, are now well resolved and rendered interpretable. Our analysis reveals the existence of three major conformations: in addition to the previously described ATP-hydrolyzing (ATP h ) and ATP-γS conformations, an intermediate state has been found. Its AAA-ATPase module adopts essentially the same topology that is observed in the ATP h conformation, whereas the lid is more similar to the ATP-γS bound state. Based on the conformational ensemble of the 26S proteasome in solution, we propose a mechanistic model for substrate recognition, commitment, deubiquitylation, and translocation into the core particle.conformational switching | proteolysis | proteostasis | quality control I n the ubiquitin-proteasome pathway (UPP) the 26S proteasome performs the degradation of intracellular proteins marked for destruction by the covalent attachment of polyubiquitin chains (1-5). The 2.5 MDa complex consists of the barrel-shaped 20S core particle (CP) as well as one or two copies of the 19S regulatory particle (RP) controlling the entry of substrates into the proteolytic chamber of the CP. The structure of the CP was solved by X-ray crystallography a long time ago (6, 7); whereas, the molecular architecture of the 26S holocomplex was determined only recently using cryo-EM single-particle analysis (SPA) approaches (8-10). The RP comprises a ringshaped AAA-ATPase heterohexamer (Rpt1-6) responsible for substrate unfolding and translocation across the CP gate and 13 RP non-ATPases (Rpn1-3, 5-13, 15) surrounding the AAAATPase module. The role of the Rpns is the acceptance of substrates and their deubiquitylation. For a full mechanistic understanding of the early steps of substrate processing it is essential to reveal its dynamics.The compositional and conformational heterogeneity of 26S proteasome preparations makes the structural characterization of this molecular machine challenging (11). Compositional heterogeneity results from multiple proteins that interact with the 26S proteasome substoichiometrically, such as deubiquitylating enzymes (DUBs) or shuttling ubiquitin (Ub) receptors. Conformational switching of the 26S proteasome is mostly driven by ATP binding and hydrolysis. Each of the six distinct Rpt subunits is able to bind and hydrolyze ATP (12-14), which may...

The 26S proteasome is a 2.5-MDa, ATP-dependent multisubunit proteolytic complex that processively destroys proteins carrying a degradation signal. The proteasomal ATPase heterohexamer is a key module of the 19S regulatory particle; it unfolds substrates and translocates them into the 20S core particle where degradation takes place. We used cryoelectron microscopy single-particle analysis to obtain insights into the structural changes of 26S proteasome upon the binding and hydrolysis of ATP. The ATPase ring adopts at least two distinct helical staircase conformations dependent on the nucleotide state. The transition from the conformation observed in the presence of ATP to the predominant conformation in the presence of ATP-γS induces a sliding motion of the ATPase ring over the 20S core particle ring leading to an alignment of the translocation channels of the ATPase and the core particle gate, a conformational state likely to facilitate substrate translocation. Two types of intersubunit modules formed by the large ATPase domain of one ATPase subunit and the small ATPase domain of its neighbor exist. They resemble the contacts observed in the crystal structures of ClpX and proteasome-activating nucleotidase, respectively. The ClpX-like contacts are positioned consecutively and give rise to helical shape in the hexamer, whereas the proteasome-activating nucleotidase-like contact is required to close the ring. Conformational switching between these forms allows adopting different helical conformations in different nucleotide states. We postulate that ATP hydrolysis by the regulatory particle ATPase (Rpt) 5 subunit initiates a cascade of conformational changes, leading to pulling of the substrate, which is primarily executed by Rpt1, Rpt2, and Rpt6.AAA ATPase | ubiquitin-proteasome pathway | hybrid methods in structural biology T he 26S proteasome is the executive arm of the ubiquitin-proteasome system (UPS), the major pathway for intracellular protein degradation in eukaryotic cells (1, 2). It degrades proteins that are marked for destruction by the covalent attachment of a polyubiquitin chain. The 2.5-MDa complex comprises a core particle (CP), the 20S proteasome, harboring the proteolytically active sites and one or two regulatory particles (RPs), which associate with the barrel-shaped CP. A key component of the RP is the AAA-ATPase module (ATPase associated with various cellular activities), which associates with the α-rings of the CP and prepares substrates for degradation. Whereas the 26S proteasome is the only soluble ATP-dependent protease in the eukaryotic cytosol, bacteria possess several different ATPdependent proteases like the ClpXP, HslUV, and the eubacterial proteasome-ARC/ Mpa (AAA ATPase forming ring-shaped complexes/mycobacterial proteasomal ATPase) system (3).The CP consists of four seven-membered rings (4). The two adjacent heteroheptameric β-rings form a cavity, which harbors the active sites (5, 6). The β-rings are sandwiched between two heteroheptameric α-rings, whose C-termini form a gate con...

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The ATP-dependent degradation of polyubiquitylated proteins by the 26S proteasome is essential for the maintenance of proteome stability and the regulation of a plethora of cellular processes. Degradation of substrates is preceded by the removal of polyubiquitin moieties through the isopeptidase activity of the subunit Rpn11. Here we describe three crystal structures of the heterodimer of the Mpr1-Pad1-N-terminal domains of Rpn8 and Rpn11, crystallized as a fusion protein in complex with a nanobody. This fusion protein exhibits modest deubiquitylation activity toward a model substrate. Full activation requires incorporation of Rpn11 into the 26S proteasome and is dependent on ATP hydrolysis, suggesting that substrate processing and polyubiquitin removal are coupled. Based on our structures, we propose that premature activation is prevented by the combined effects of low intrinsic ubiquitin affinity, an insertion segment acting as a physical barrier across the substrate access channel, and a conformationally unstable catalytic loop in Rpn11. The docking of the structure into the proteasome EM density revealed contacts of Rpn11 with ATPase subunits, which likely stabilize the active conformation and boost the affinity for the proximal ubiquitin moiety. The narrow space around the Rpn11 active site at the entrance to the ATPase ring pore is likely to prevent erroneous deubiquitylation of folded proteins.n eukaryotes, the ubiquitin (Ub) proteasome system (UPS) is responsible for the regulated degradation of proteins (1-5). The UPS plays a key role in the maintenance of protein homeostasis by removing misfolded or damaged proteins, which could impair cellular functions, and by removing proteins whose functions are no longer needed. Consequently, the UPS is critically involved in numerous cellular processes, including cell cycle progression, apoptosis, and DNA damage repair, and malfunctions of the system often result in disease.The 26S proteasome executes the degradation of substrates that are marked for destruction by the covalent attachment of polyubiquitin chains. It is a molecular machine of 2.5 MDa comprising two subcomplexes, the 20S core particle (CP) and one or two 19S regulatory particles (RPs), which associate with the ends of the cylinder-shaped CP (6-8). The recognition and recruitment of polyubiquitylated substrates, their deubiquitylation, ATP-dependent unfolding, and translocation into the core particle take place in the RP. The structurally and mechanistically well-characterized CP houses the proteolytic activities and sequesters them from the environment, thereby avoiding collateral damage (9).The RPs attach to the outer α-rings of the CP, which control access to the proteolytic chamber formed by the inner β-subunit rings (10). Recently, the molecular architecture of the 26S holocomplex was established using cryo-EM-based approaches (11,12), and a pseudoatomic model of the holocomplex was put forward (13). The RP is formed by two subcomplexes, known as the base and the lid, which assemble independent...