Targeted protein degradation via intramolecular bivalent glues.

Targeted protein degradation is a pharmacological modality that is based on the induced proximity of an E3 ubiquitin ligase and a target protein to promote target ubiquitination and proteasomal degradation. This has been achieved either via proteolysis-targeting chimeras (PROTACs)-bifunctional compounds composed of two separate moieties that individually bind the target and E3 ligase, or via molecular glues that monovalently bind either the ligase or the target. Here, using orthogonal genetic screening, biophysical characterization and structural reconstitution, we investigate the mechanism of action of bifunctional degraders of BRD2 and BRD4, termed intramolecular bivalent glues (IBGs), and find that instead of connecting target and ligase in trans as PROTACs do, they simultaneously engage and connect two adjacent domains of the target protein in cis.

RNA polymerase II promotes the organization of chromatin following DNA replication.

Understanding how chromatin organisation is duplicated on the two daughter strands is a central question in epigenetics. In mammals, following the passage of the replisome, nucleosomes lose their defined positioning and transcription contributes to their re-organisation. However, whether transcription plays a greater role in the organization of chromatin following DNA replication remains unclear.

Structure and mechanism of the type I-G CRISPR effector.

Type I CRISPR systems are the most common CRISPR type found in bacteria. They use a multisubunit effector, guided by crRNA, to detect and bind dsDNA targets, forming an R-loop and recruiting the Cas3 enzyme to facilitate target DNA destruction, thus providing immunity against mobile genetic elements. Subtypes have been classified into families A-G, with type I-G being the least well understood.

A non-canonical scaffold-type E3 ligase complex mediates protein UFMylation.

Protein UFMylation, i.e., post-translational modification with ubiquitin-fold modifier 1 (UFM1), is essential for cellular and endoplasmic reticulum homeostasis.

Acute depletion of the ARID1A subunit of SWI/SNF complexes reveals distinct pathways for activation and repression of transcription.

The ARID1A subunit of SWI/SNF chromatin remodeling complexes is a potent tumor suppressor. Here, a degron is applied to detect rapid loss of chromatin accessibility at thousands of loci where ARID1A acts to generate accessible minidomains of nucleosomes. Loss of ARID1A also results in the redistribution of the coactivator EP300.

Efficient and Rapid Analysis of Polysomes and Ribosomal Subunits in Cells and Tissues Using Ribo Mega-SEC.

Polysome profile analysis is a popular method for separating polysomes and ribosomal subunits and is typically achieved using a sucrose density gradient (SDG). This has remained the gold standard method since ribosomes were first discovered; however, this method is time-consuming and requires multiple steps from making the gradient and long ultracentrifugation to collecting and analyzing the fractions. Each of these steps in the SDG workflow can introduce potential technical variation that affects the reproducibility of gradient profiles between samples.

Nucleosome remodelling: structural insights into ATP-dependent remodelling enzymes.

ATP-dependent chromatin remodelling enzymes play a fundamental role in determining how nucleosomes are organised, and render DNA sequences accessible to interacting proteins, thereby enabling precise regulation of eukaryotic genes. Remodelers conserved from yeast to humans are classified into four families based on the domains and motifs present in their ATPase subunits. Insights into overall assembly and the mode of interaction to the nucleosome by these different families of remodelers remained limited due to the complexity of obtaining structural information on these challenging samples.

Efficient analysis of mammalian polysomes in cells and tissues using Ribo Mega-SEC.

We describe Ribo Mega-SEC, a powerful approach for the separation and biochemical analysis of mammalian polysomes and ribosomal subunits using Size Exclusion Chromatography and uHPLC. Using extracts from either cells, or tissues, polysomes can be separated within 15 min from sample injection to fraction collection. Ribo Mega-SEC shows translating ribosomes exist predominantly in polysome complexes in human cell lines and mouse liver tissue.

Structure of the chromatin remodelling enzyme Chd1 bound to a ubiquitinylated nucleosome.

ATP-dependent chromatin remodelling proteins represent a diverse family of proteins that share ATPase domains that are adapted to regulate protein-DNA interactions. Here, we present structures of the Chd1 protein engaged with nucleosomes in the presence of the transition state mimic ADP-beryllium fluoride. The path of DNA strands through the ATPase domains indicates the presence of contacts conserved with single strand translocases and additional contacts with both strands that are unique to Snf2 related proteins.

Activity-based E3 ligase profiling uncovers an E3 ligase with esterification activity.

Ubiquitination is initiated by transfer of ubiquitin (Ub) from a ubiquitin-activating enzyme (E1) to a ubiquitin-conjugating enzyme (E2), producing a covalently linked intermediate (E2-Ub) . Ubiquitin ligases (E3s) of the 'really interesting new gene' (RING) class recruit E2-Ub via their RING domain and then mediate direct transfer of ubiquitin to substrates . By contrast, 'homologous to E6-AP carboxy terminus' (HECT) E3 ligases undergo a catalytic cysteine-dependent transthiolation reaction with E2-Ub, forming a covalent E3-Ub intermediate.

The Ndc80 complex targets Bod1 to human mitotic kinetochores.

Regulation of protein phosphatase activity by endogenous protein inhibitors is an important mechanism to control protein phosphorylation in cells. We recently identified Biorientation defective 1 (Bod1) as a small protein inhibitor of protein phosphatase 2A containing the B56 regulatory subunit (PP2A-B56). This phosphatase controls the amount of phosphorylation of several kinetochore proteins and thus the establishment of load-bearing chromosome-spindle attachments in time for accurate separation of sister chromatids in mitosis.

Parkin-phosphoubiquitin complex reveals cryptic ubiquitin-binding site required for RBR ligase activity.

RING-between-RING (RBR) E3 ligases are a class of ubiquitin ligases distinct from RING or HECT E3 ligases. An important RBR ligase is Parkin, mutations in which lead to early-onset hereditary Parkinsonism. Parkin and other RBR ligases share a catalytic RBR module but are usually autoinhibited and activated via distinct mechanisms.

Structural reorganization of the chromatin remodeling enzyme Chd1 upon engagement with nucleosomes.

The yeast Chd1 protein acts to position nucleosomes across genomes. Here, we model the structure of the Chd1 protein in solution and when bound to nucleosomes. In the apo state, the DNA-binding domain contacts the edge of the nucleosome while in the presence of the non-hydrolyzable ATP analog, ADP-beryllium fluoride, we observe additional interactions between the ATPase domain and the adjacent DNA gyre 1.

The histone chaperone Vps75 forms multiple oligomeric assemblies capable of mediating exchange between histone H3-H4 tetramers and Asf1-H3-H4 complexes.

Vps75 is a histone chaperone that has been historically characterized as homodimer by X-ray crystallography. In this study, we present a crystal structure containing two related tetrameric forms of Vps75 within the crystal lattice. We show Vps75 associates with histones in multiple oligomers.

Disruption of the autoinhibited state primes the E3 ligase parkin for activation and catalysis.

The PARK2 gene is mutated in 50% of autosomal recessive juvenile parkinsonism (ARJP) cases. It encodes parkin, an E3 ubiquitin ligase of the RBR family. Parkin exists in an autoinhibited state that is activated by phosphorylation of its N-terminal ubiquitin-like (Ubl) domain and binding of phosphoubiquitin.

Mechanisms and functions of ATP-dependent chromatin-remodeling enzymes.

Chromatin provides both a means to accommodate a large amount of genetic material in a small space and a means to package the same genetic material in different chromatin states. Transitions between chromatin states are enabled by chromatin-remodeling ATPases, which catalyze a diverse range of structural transformations. Biochemical evidence over the last two decades suggests that chromatin-remodeling activities may have emerged by adaptation of ancient DNA translocases to respond to specific features of chromatin.

The DNA-binding domain of the Chd1 chromatin-remodelling enzyme contains SANT and SLIDE domains.

The ATP-dependent chromatin-remodelling enzyme Chd1 is a 168-kDa protein consisting of a double chromodomain, Snf2-related ATPase domain, and a C-terminal DNA-binding domain. Here, we show the DNA-binding domain is required for Saccharomyces cerevisiae Chd1 to bind and remodel nucleosomes. The crystal structure of this domain reveals the presence of structural homology to SANT and SLIDE domains previously identified in ISWI remodelling enzymes.

Structure of Staphylococcus aureus EsxA suggests a contribution to virulence by action as a transport chaperone and/or adaptor protein.

Staphylococcus aureus pathogenesis depends on a specialized protein secretion system (ESX-1) that delivers a range of virulence factors to assist infectivity. We report the characterization of two such factors, EsxA and EsxB, small acidic dimeric proteins carrying a distinctive WXG motif. EsxA crystallized in triclinic and monoclinic forms and high-resolution structures were determined.

Crystal structures of a bacterial 6-phosphogluconate dehydrogenase reveal aspects of specificity, mechanism and mode of inhibition by analogues of high-energy reaction intermediates.

Crystal structures of recombinant Lactococcus lactis 6-phosphogluconate dehydrogenase (LlPDH) in complex with substrate, cofactor, product and inhibitors have been determined. LlPDH shares significant sequence identity with the enzymes from sheep liver and the protozoan parasite Trypanosoma brucei for which structures have been reported. Comparisons indicate that the key residues in the active site are highly conserved, as are the interactions with the cofactor and the product ribulose 5-phosphate.

The crystal structure of a plant 3-ketoacyl-CoA thiolase reveals the potential for redox control of peroxisomal fatty acid beta-oxidation.

Crystal structures of peroxisomal Arabidopsis thaliana 3-ketoacyl-CoA thiolase (AtKAT), an enzyme of fatty acid beta-oxidation, are reported. The subunit, a typical thiolase, is a combination of two similar alpha/beta domains capped with a loop domain. The comparison of AtKAT with the Saccharomyces cerevisiae homologue (ScKAT) structure reveals a different placement of subunits within the functional dimers and that a polypeptide segment forming an extended loop around the open catalytic pocket of ScKAT converts to alpha-helix in AtKAT, and occludes the active site.

Effect of dimethyl sulphoxide on the crystal structure of porcine pepsin.

The structure of porcine pepsin crystallized in the presence of dimethyl sulphoxide has been analysed by X-ray crystallography to obtain insights into the structural events that occur at the onset of chemical denaturation of proteins. The results show that one dimethyl sulphoxide molecule occupies a site on the surface of pepsin interacting with two of its residues. An increase in the average temperature factor of pepsin in the presence of dimethyl sulphoxide has been observed indicating protein destabilization induced by the denaturant.