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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.

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.

Methylation of adenosines at the N(6) position (m(6)A) 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 m(6)A 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 m(6)A 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 m(6)A modification in post-transcriptional genome regulation.

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.

This protocol describes the screening of a library of low-molecular-weight compounds (fragments) using a series of biophysical ligand-binding assays. Fragment-based drug discovery (FBDD) has emerged as a successful method to design high-affinity ligands for biomacromolecules of therapeutic interest. It involves detecting relatively weak interactions between the fragments and a target macromolecule using sensitive biophysical techniques. These weak binders provide a starting point for the development of inhibitors with submicromolar affinity. Here we describe an efficient fragment screening cascade that can identify binding fragments (hits) within weeks. It is divided into three stages: (i) preliminary screening using differential scanning fluorimetry (DSF), (ii) validation by NMR spectroscopy and (iii) characterization of binding fragments by isothermal titration calorimetry (ITC) and X-ray crystallography. Although this protocol is readily applicable in academic settings because of its emphasis on low cost and medium-throughput early-stage screening technologies, the core principle of orthogonal validation makes it robust enough to meet the quality standards of an industrial laboratory.

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.

Despite intensive effort, the majority of the annotated Mycobacterium tuberculosis genome consists of genes encoding proteins of unknown or poorly understood function. For example, there are seven conserved hypothetical proteins annotated as homologs of pyridoxine 5'-phosphate oxidase (PNPOx), an enzyme that oxidizes pyridoxine 5'-phosphate (PNP) or pyridoxamine 5'-phosphate (PMP) to form pyridoxal 5'-phosphate (PLP). We have characterized the function of Rv2607 from Mycobacterium tuberculosis H37Rv and shown that it encodes a PNPOx that oxidizes PNP to PLP. The k(cat) and K(M) for this reaction were 0.01 s(-1) and 360 µM, respectively. Unlike many PNPOx enzymes, Rv2607 does not recognize PMP as a substrate.

One‐dimensional 1 H NMR experiments are commonly employed in both industry and academia for fragment‐based screening against biomolecular protein targets. The overall objective of this chapter is to familiarize the reader with the principles of ligand‐based NMR binding experiments, including an understanding of the theory behind each experiment, and to describe in detail the application of these methods to fragment screening and hit validation. First, some of the theoretical principles of ligand‐observed NMR techniques and their advantages relative to protein‐observed NMR methods are briefly illustrated. We then describe how NMR experiments are used to obtain: (i) yes/no binding answers, (ii) measurements of relative affinities, and (iii) information on binding modes. Finally, practical considerations on performing actual experiments, and processing and analyzing the data are highlighted. The chapter focuses on NMR sequences employing relaxation‐edited filters, saturation transfer difference (STD), water‐ligand observed by gradient spectroscopy (WaterLOGSY), and the interligand Overhauser effect (ILOE), which are mainly employed in our laboratories. Other related ligand‐observed NMR approaches are also described briefly. We especially emphasize our experiences and lessons learnt over the past few years on implementing and applying these techniques to fragment screening and fragment‐based discovery efforts in an academic setup.

Methylation of adenosines at the N.sup.6 position (m.sup.6A) 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 m.sup.6A 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 m.sup.6A 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 m.sup.6A modification in post-transcriptional genome regulation.