Positive selection CRISPR screens reveal a druggable pocket in an oligosaccharyltransferase required for inflammatory signaling to NF-κB.

Nuclear factor κB (NF-κB) plays roles in various diseases. Many inflammatory signals, such as circulating lipopolysaccharides (LPSs), activate NF-κB via specific receptors. Using whole-genome CRISPR-Cas9 screens of LPS-treated cells that express an NF-κB-driven suicide gene, we discovered that the LPS receptor Toll-like receptor 4 (TLR4) is specifically dependent on the oligosaccharyltransferase complex OST-A for N-glycosylation and cell-surface localization. The tool compound NGI-1 inhibits OST complexes in vivo, but the underlying molecular mechanism remained unknown. We did a CRISPR base-editor screen for NGI-1-resistant variants of STT3A, the catalytic subunit of OST-A. These variants, in conjunction with cryoelectron microscopy studies, revealed that NGI-1 binds the catalytic site of STT3A, where it traps a molecule of the donor substrate dolichyl-PP-GlcNAc2-Man9-Glc3, suggesting an uncompetitive inhibition mechanism. Our results provide a rationale for and an initial step toward the development of STT3A-specific inhibitors and illustrate the power of contemporaneous base-editor and structural studies to define drug mechanism of action.

Thiolase: A Versatile Biocatalyst Employing Coenzyme A-Thioester Chemistry for Making and Breaking C-C Bonds.

Thiolases are CoA-dependent enzymes that catalyze the thiolytic cleavage of 3-ketoacyl-CoA, as well as its reverse reaction, which is the thioester-dependent Claisen condensation reaction. Thiolases are dimers or tetramers (dimers of dimers). All thiolases have two reactive cysteines: (a) a nucleophilic cysteine, which forms a covalent intermediate, and (b) an acid/base cysteine. The best characterized thiolase is the Zoogloea ramigera thiolase, which is a bacterial biosynthetic thiolase belonging to the CT-thiolase subfamily. The thiolase active site is also characterized by two oxyanion holes, two active site waters, and four catalytic loops with characteristic amino acid sequence fingerprints. Three thiolase subfamilies can be identified, each characterized by a unique sequence fingerprint for one of their catalytic loops, which causes unique active site properties. Recent insights concerning the thiolase reaction mechanism, as obtained from recent structural studies, as well as from classical and recent enzymological studies, are addressed, and open questions are discussed.

Reconstruction of a fatty acid synthesis cycle from acyl carrier protein and cofactor structural snapshots.

Fatty acids (FAs) play a central metabolic role in living cells as constituents of membranes, cellular energy reserves, and second messenger precursors. A 2.6 MDa FA synthase (FAS), where the enzymatic reactions and structures are known, is responsible for FA biosynthesis in yeast. Essential in the yeast FAS catalytic cycle is the acyl carrier protein (ACP) that actively shuttles substrates, biosynthetic intermediates, and products from one active site to another. We resolve the S. cerevisiae FAS structure at 1.9 Å, elucidating cofactors and water networks involved in their recognition. Structural snapshots of ACP domains bound to various enzymatic domains allow the reconstruction of a full yeast FA biosynthesis cycle. The structural information suggests that each FAS functional unit could accommodate exogenous proteins to incorporate various enzymatic activities, and we show proof-of-concept experiments where ectopic proteins are used to modulate FAS product profiles.

Massively parallel assessment of human variants with base editor screens.

Understanding the functional consequences of single-nucleotide variants is critical to uncovering the genetic underpinnings of diseases, but technologies to characterize variants are limiting. Here, we leverage CRISPR-Cas9 cytosine base editors in pooled screens to scalably assay variants at endogenous loci in mammalian cells. We benchmark the performance of base editors in positive and negative selection screens, identifying known loss-of-function mutations in BRCA1 and BRCA2 with high precision. To demonstrate the utility of base editor screens to probe small molecule-protein interactions, we screen against BH3 mimetics and PARP inhibitors, identifying point mutations that confer drug sensitivity or resistance. We also create a library of single guide RNAs (sgRNAs) predicted to generate 52,034 ClinVar variants in 3,584 genes and conduct screens in the presence of cellular stressors, identifying loss-of-function variants in numerous DNA damage repair genes. We anticipate that this screening approach will be broadly useful to readily and scalably functionalize genetic variants.

RNA Splicing by the Spliceosome.

The spliceosome removes introns from messenger RNA precursors (pre-mRNA). Decades of biochemistry and genetics combined with recent structural studies of the spliceosome have produced a detailed view of the mechanism of splicing. In this review, we aim to make this mechanism understandable and provide several videos of the spliceosome in action to illustrate the intricate choreography of splicing. The U1 and U2 small nuclear ribonucleoproteins (snRNPs) mark an intron and recruit the U4/U6.U5 tri-snRNP. Transfer of the 5' splice site (5'SS) from U1 to U6 snRNA triggers unwinding of U6 snRNA from U4 snRNA. U6 folds with U2 snRNA into an RNA-based active site that positions the 5'SS at two catalytic metal ions. The branch point (BP) adenosine attacks the 5'SS, producing a free 5' exon. Removal of the BP adenosine from the active site allows the 3'SS to bind, so that the 5' exon attacks the 3'SS to produce mature mRNA and an excised lariat intron.

How Is Precursor Messenger RNA Spliced by the Spliceosome?

Splicing of the precursor messenger RNA, involving intron removal and exon ligation, is mediated by the spliceosome. Together with biochemical and genetic investigations of the past four decades, structural studies of the intact spliceosome at atomic resolution since 2015 have led to mechanistic delineation of RNA splicing with remarkable insights. The spliceosome is proven to be a protein-orchestrated metalloribozyme. Conserved elements of small nuclear RNA (snRNA) constitute the splicing active site with two catalytic metal ions and recognize three conserved intron elements through duplex formation, which are delivered into the splicing active site for branching and exon ligation. The protein components of the spliceosome stabilize the conformation of the snRNA, drive spliceosome remodeling, orchestrate the movement of the RNA elements, and facilitate the splicing reaction. The overall organization of the spliceosome and the configuration of the splicing active site are strictly conserved between human and yeast.

Signaling by the phosphoinositide 3-kinase family in immune cells.

Phosphoinositide 3-kinases (PI3Ks) control many important aspects of immune cell development, differentiation, and function. Mammals have eight PI3K catalytic subunits that are divided into three classes based on similarities in structure and function. Specific roles for the class I PI3Ks have been broadly investigated and are relatively well understood, as is the function of their corresponding phosphatases. More recently, specific roles for the class II and class III PI3Ks have emerged. Through vertebrate evolution and in parallel with the evolution of adaptive immunity, there has been a dramatic increase not only in the genes for PI3K subunits but also in genes for phosphatases that act on 3-phosphoinositides and in 3-phosphoinositide-binding proteins. Our understanding of the PI3Ks in immunity is guided by fundamental discoveries made in simpler model organisms as well as by appreciating new adaptations of this signaling module in mammals in general and in immune cells in particular.

Dual Functions of a Rubisco Activase in Metabolic Repair and Recruitment to Carboxysomes.

Rubisco, the key enzyme of CO2 fixation in photosynthesis, is prone to inactivation by inhibitory sugar phosphates. Inhibited Rubisco undergoes conformational repair by the hexameric AAA+ chaperone Rubisco activase (Rca) in a process that is not well understood. Here, we performed a structural and mechanistic analysis of cyanobacterial Rca, a close homolog of plant Rca. In the Rca:Rubisco complex, Rca is positioned over the Rubisco catalytic site under repair and pulls the N-terminal tail of the large Rubisco subunit (RbcL) into the hexamer pore. Simultaneous displacement of the C terminus of the adjacent RbcL opens the catalytic site for inhibitor release. An alternative interaction of Rca with Rubisco is mediated by C-terminal domains that resemble the small Rubisco subunit. These domains, together with the N-terminal AAA+ hexamer, ensure that Rca is packaged with Rubisco into carboxysomes. The cyanobacterial Rca is a dual-purpose protein with functions in Rubisco repair and carboxysome organization.

Discovery of a Regulatory Subunit of the Yeast Fatty Acid Synthase.

Fatty acid synthases (FASs) are central to metabolism but are also of biotechnological interest for the production of fine chemicals and biofuels from renewable resources. During fatty acid synthesis, the growing fatty acid chain is thought to be shuttled by the dynamic acyl carrier protein domain to several enzyme active sites. Here, we report the discovery of a γ subunit of the 2.6 megadalton α6-ß6S. cerevisiae FAS, which is shown by high-resolution structures to stabilize a rotated FAS conformation and rearrange ACP domains from equatorial to axial positions. The γ subunit spans the length of the FAS inner cavity, impeding reductase activities of FAS, regulating NADPH turnover by kinetic hysteresis at the ketoreductase, and suppressing off-pathway reactions at the enoylreductase. The γ subunit delineates the functional compartment within FAS. As a scaffold, it may be exploited to incorporate natural and designed enzymatic activities that are not present in natural FAS.

A Structure-Based Model for the Complete Transcription Cycle of Influenza Polymerase.

Influenza polymerase uses unique mechanisms to synthesize capped and polyadenylated mRNAs from the genomic viral RNA (vRNA) template, which is packaged inside ribonucleoprotein particles (vRNPs). Here, we visualize by cryoelectron microscopy the conformational dynamics of the polymerase during the complete transcription cycle from pre-initiation to termination, focusing on the template trajectory. After exiting the active site cavity, the template 3' extremity rebinds into a specific site on the polymerase surface. Here, it remains sequestered during all subsequent transcription steps, forcing the template to loop out as it further translocates. At termination, the strained connection between the bound template 5' end and the active site results in polyadenylation by stuttering at uridine 17. Upon product dissociation, further conformational changes release the trapped template, allowing recycling back into the pre-initiation state. Influenza polymerase thus performs transcription while tightly binding to and protecting both template ends, allowing efficient production of multiple mRNAs from a single vRNP.

Structural Basis for RNA Replication by the SARS-CoV-2 Polymerase.

Nucleotide analog inhibitors, including broad-spectrum remdesivir and favipiravir, have shown promise in in vitro assays and some clinical studies for COVID-19 treatment, this despite an incomplete mechanistic understanding of the viral RNA-dependent RNA polymerase nsp12 drug interactions. Here, we examine the molecular basis of SARS-CoV-2 RNA replication by determining the cryo-EM structures of the stalled pre- and post- translocated polymerase complexes. Compared with the apo complex, the structures show notable structural rearrangements happening to nsp12 and its co-factors nsp7 and nsp8 to accommodate the nucleic acid, whereas there are highly conserved residues in nsp12, positioning the template and primer for an in-line attack on the incoming nucleotide. Furthermore, we investigate the inhibition mechanism of the triphosphate metabolite of remdesivir through structural and kinetic analyses. A transition model from the nsp7-nsp8 hexadecameric primase complex to the nsp12-nsp7-nsp8 polymerase complex is also proposed to provide clues for the understanding of the coronavirus transcription and replication machinery.

Cryo-EM Structures of a Group II Intron Reverse Splicing into DNA.

Group II introns are a class of retroelements that invade DNA through a copy-and-paste mechanism known as retrotransposition. Their coordinated activities occur within a complex that includes a maturase protein, which promotes splicing through an unknown mechanism. The mechanism of splice site exchange within the RNA active site during catalysis also remains unclear. We determined two cryo-EM structures at 3.6-Å resolution of a group II intron reverse splicing into DNA. These structures reveal that the branch-site domain VI helix swings 90°, enabling substrate exchange during DNA integration. The maturase assists catalysis through a transient RNA-protein contact with domain VI that positions the branch-site adenosine for lariat formation during forward splicing. These findings provide the first direct evidence of the role the maturase plays during group II intron catalysis. The domain VI dynamics closely parallel spliceosomal branch-site helix movement and provide strong evidence for a retroelement origin of the spliceosome.

Mechanism of Cross-talk between H2B Ubiquitination and H3 Methylation by Dot1L.

Methylation of histone H3 K79 by Dot1L is a hallmark of actively transcribed genes that depends on monoubiquitination of H2B K120 (H2B-Ub) and is an example of histone modification cross-talk that is conserved from yeast to humans. We report here cryo-EM structures of Dot1L bound to ubiquitinated nucleosome that show how H2B-Ub stimulates Dot1L activity and reveal a role for the histone H4 tail in positioning Dot1L. We find that contacts mediated by Dot1L and the H4 tail induce a conformational change in the globular core of histone H3 that reorients K79 from an inaccessible position, thus enabling this side chain to insert into the active site in a position primed for catalysis. Our study provides a comprehensive mechanism of cross-talk between histone ubiquitination and methylation and reveals structural plasticity in histones that makes it possible for histone-modifying enzymes to access residues within the nucleosome core.

Structures of the Catalytically Activated Yeast Spliceosome Reveal the Mechanism of Branching.

Pre-mRNA splicing is executed by the spliceosome. Structural characterization of the catalytically activated complex (B∗) is pivotal for understanding the branching reaction. In this study, we assembled the B∗ complexes on two different pre-mRNAs from Saccharomyces cerevisiae and determined the cryo-EM structures of four distinct B∗ complexes at overall resolutions of 2.9-3.8 Å. The duplex between U2 small nuclear RNA (snRNA) and the branch point sequence (BPS) is discretely away from the 5'-splice site (5'SS) in the three B∗ complexes that are devoid of the step I splicing factors Yju2 and Cwc25. Recruitment of Yju2 into the active site brings the U2/BPS duplex into the vicinity of 5'SS, with the BPS nucleophile positioned 4 Å away from the catalytic metal M2. This analysis reveals the functional mechanism of Yju2 and Cwc25 in branching. These structures on different pre-mRNAs reveal substrate-specific conformations of the spliceosome in a major functional state.

Structure of the Respiratory Syncytial Virus Polymerase Complex.

Numerous interventions are in clinical development for respiratory syncytial virus (RSV) infection, including small molecules that target viral transcription and replication. These processes are catalyzed by a complex comprising the RNA-dependent RNA polymerase (L) and the tetrameric phosphoprotein (P). RSV P recruits multiple proteins to the polymerase complex and, with the exception of its oligomerization domain, is thought to be intrinsically disordered. Despite their critical roles in RSV transcription and replication, structures of L and P have remained elusive. Here, we describe the 3.2-Å cryo-EM structure of RSV L bound to tetrameric P. The structure reveals a striking tentacular arrangement of P, with each of the four monomers adopting a distinct conformation. The structure also rationalizes inhibitor escape mutants and mutations observed in live-attenuated vaccine candidates. These results provide a framework for determining the molecular underpinnings of RSV replication and transcription and should facilitate the design of effective RSV inhibitors.

Understanding and Improving the Activity of Flavin-Dependent Halogenases via Random and Targeted Mutagenesis.

Flavin-dependent halogenases (FDHs) catalyze the halogenation of organic substrates by coordinating reactions of reduced flavin, molecular oxygen, and chloride. Targeted and random mutagenesis of these enzymes have been used to both understand and alter their reactivity. These studies have led to insights into residues essential for catalysis and FDH variants with improved stability, expanded substrate scope, and altered site selectivity. Mutations throughout FDH structures have contributed to all of these advances. More recent studies have sought to rationalize the impact of these mutations on FDH function and to identify new FDHs to deepen our understanding of this enzyme class and to expand their utility for biocatalytic applications.

Protein AMPylation by an Evolutionarily Conserved Pseudokinase.

Approximately 10% of human protein kinases are believed to be inactive and named pseudokinases because they lack residues required for catalysis. Here, we show that the highly conserved pseudokinase selenoprotein-O (SelO) transfers AMP from ATP to Ser, Thr, and Tyr residues on protein substrates (AMPylation), uncovering a previously unrecognized activity for a member of the protein kinase superfamily. The crystal structure of a SelO homolog reveals a protein kinase-like fold with ATP flipped in the active site, thus providing a structural basis for catalysis. SelO pseudokinases localize to the mitochondria and AMPylate proteins involved in redox homeostasis. Consequently, SelO activity is necessary for the proper cellular response to oxidative stress. Our results suggest that AMPylation may be a more widespread post-translational modification than previously appreciated and that pseudokinases should be analyzed for alternative transferase activities.

Crystal Structure of the COMPASS H3K4 Methyltransferase Catalytic Module.

The SET1/MLL family of histone methyltransferases is conserved in eukaryotes and regulates transcription by catalyzing histone H3K4 mono-, di-, and tri-methylation. These enzymes form a common five-subunit catalytic core whose assembly is critical for their basal and regulated enzymatic activities through unknown mechanisms. Here, we present the crystal structure of the intact yeast COMPASS histone methyltransferase catalytic module consisting of Swd1, Swd3, Bre2, Sdc1, and Set1. The complex is organized by Swd1, whose conserved C-terminal tail not only nucleates Swd3 and a Bre2-Sdc1 subcomplex, but also joins Set1 to construct a regulatory pocket next to the catalytic site. This inter-subunit pocket is targeted by a previously unrecognized enzyme-modulating motif in Swd3 and features a doorstop-style mechanism dictating substrate selectivity among SET1/MLL family members. By spatially mapping the functional components of COMPASS, our results provide a structural framework for understanding the multifaceted functions and regulation of the H3K4 methyltransferase family.

Transposase-DNA Complex Structures Reveal Mechanisms for Conjugative Transposition of Antibiotic Resistance.

Conjugative transposition drives the emergence of multidrug resistance in diverse bacterial pathogens, yet the mechanisms are poorly characterized. The Tn1549 conjugative transposon propagates resistance to the antibiotic vancomycin used for severe drug-resistant infections. Here, we present four high-resolution structures of the conserved Y-transposase of Tn1549 complexed with circular transposon DNA intermediates. The structures reveal individual transposition steps and explain how specific DNA distortion and cleavage mechanisms enable DNA strand exchange with an absolute minimum homology requirement. This appears to uniquely allow Tn916-like conjugative transposons to bypass DNA homology and insert into diverse genomic sites, expanding gene transfer. We further uncover a structural regulatory mechanism that prevents premature cleavage of the transposon DNA before a suitable target DNA is found and generate a peptide antagonist that interferes with the transposase-DNA structure to block transposition. Our results reveal mechanistic principles of conjugative transposition that could help control the spread of antibiotic resistance genes.

Structure and Conformational Dynamics of a COMPASS Histone H3K4 Methyltransferase Complex.

The methylation of histone 3 lysine 4 (H3K4) is carried out by an evolutionarily conserved family of methyltransferases referred to as complex of proteins associated with Set1 (COMPASS). The activity of the catalytic SET domain (su(var)3-9, enhancer-of-zeste, and trithorax) is endowed through forming a complex with a set of core proteins that are widely shared from yeast to humans. We obtained cryo-electron microscopy (cryo-EM) maps of the yeast Set1/COMPASS core complex at overall 4.0- to 4.4-Å resolution, providing insights into its structural organization and conformational dynamics. The Cps50 C-terminal tail weaves within the complex to provide a central scaffold for assembly. The SET domain, snugly positioned at the junction of the Y-shaped complex, is extensively contacted by Cps60 (Bre2), Cps50 (Swd1), and Cps30 (Swd3). The mobile SET-I motif of the SET domain is engaged by Cps30, explaining its key role in COMPASS catalytic activity toward higher H3K4 methylation states.