Cryo-EM Structure of the Fork Protection Complex Bound to CMG at a Replication Fork.

The eukaryotic replisome, organized around the Cdc45-MCM-GINS (CMG) helicase, orchestrates chromosome replication. Multiple factors associate directly with CMG, including Ctf4 and the heterotrimeric fork protection complex (Csm3/Tof1 and Mrc1), which has important roles including aiding normal replication rates and stabilizing stalled forks. How these proteins interface with CMG to execute these functions is poorly understood. Here we present 3 to 3.5 Å resolution electron cryomicroscopy (cryo-EM) structures comprising CMG, Ctf4, and the fork protection complex at a replication fork. The structures provide high-resolution views of CMG-DNA interactions, revealing a mechanism for strand separation, and show Csm3/Tof1 "grip" duplex DNA ahead of CMG via a network of interactions important for efficient replication fork pausing. Although Mrc1 was not resolved in our structures, we determine its topology in the replisome by cross-linking mass spectrometry. Collectively, our work reveals how four highly conserved replisome components collaborate with CMG to facilitate replisome progression and maintain genome stability.

A conserved mechanism for regulating replisome disassembly in eukaryotes.

Replisome disassembly is the final step of eukaryotic DNA replication and is triggered by ubiquitylation of the CDC45-MCM-GINS (CMG) replicative helicase1-3. Despite being driven by evolutionarily diverse E3 ubiquitin ligases in different eukaryotes (SCFDia2 in budding yeast1, CUL2LRR1 in metazoa4-7), replisome disassembly is governed by a common regulatory principle, in which ubiquitylation of CMG is suppressed before replication termination, to prevent replication fork collapse. Recent evidence suggests that this suppression is mediated by replication fork DNA8-10. However, it is unknown how SCFDia2 and CUL2LRR1 discriminate terminated from elongating replisomes, to selectively ubiquitylate CMG only after termination. Here we used cryo-electron microscopy to solve high-resolution structures of budding yeast and human replisome-E3 ligase assemblies. Our structures show that the leucine-rich repeat domains of Dia2 and LRR1 are structurally distinct, but bind to a common site on CMG, including the MCM3 and MCM5 zinc-finger domains. The LRR-MCM interaction is essential for replisome disassembly and, crucially, is occluded by the excluded DNA strand at replication forks, establishing the structural basis for the suppression of CMG ubiquitylation before termination. Our results elucidate a conserved mechanism for the regulation of replisome disassembly in eukaryotes, and reveal a previously unanticipated role for DNA in preserving replisome integrity.

Structure determination by cryoEM at 100 keV.

Electron cryomicroscopy can, in principle, determine the structures of most biological molecules but is currently limited by access, specimen preparation difficulties, and cost. We describe a purpose-built instrument operating at 100 keV-including advances in electron optics, detection, and processing-that makes structure determination fast and simple at a fraction of current costs. The instrument attains its theoretical performance limits, allowing atomic resolution imaging of gold test specimens and biological molecular structure determination in hours. We demonstrate its capabilities by determining the structures of eleven different specimens, ranging in size from 140 kDa to 2 MDa, using a fraction of the data normally required. CryoEM with a microscope designed specifically for high-efficiency, on-the-spot imaging of biological molecules will expand structural biology to a wide range of previously intractable problems.

Smart data collection for CryoEM.

This report provides an overview of the discussions, presentations, and consensus thinking from the Workshop on Smart Data Collection for CryoEM held at the New York Structural Biology Center on April 6-7, 2022. The goal of the workshop was to address next generation data collection strategies that integrate machine learning and real-time processing into the workflow to reduce or eliminate the need for operator intervention.

A multipronged approach to understanding the form and function of hStaufen protein.

Staufen is a dsRNA-binding protein involved in many aspects of RNA regulation, such as mRNA transport, Staufen-mediated mRNA decay and the regulation of mRNA translation. It is a modular protein characterized by the presence of conserved consensus amino acid sequences that fold into double-stranded RNA binding domains (RBDs) as well as degenerated RBDs that are instead involved in protein-protein interactions. The variety of biological processes in which Staufen participates in the cell suggests that this protein associates with many diverse RNA targets, some of which have been identified experimentally. Staufen binding mediates the recruitment of effectors via protein-protein and protein-RNA interactions. The structural determinants of a number of these interactions, as well as the structure of full-length Staufen, remain unknown. Here, we present the first solution structure models for full-length hStaufen155, showing that its domains are arranged as beads-on-a-string connected by flexible linkers. In analogy with other nucleic acid-binding proteins, this could underpin Stau1 functional plasticity.

Structure of the CRISPR interference complex CSM reveals key similarities with cascade.

The Clustered Regularly Interspaced Palindromic Repeats (CRISPR) system is an adaptive immune system in prokaryotes. Interference complexes encoded by CRISPR-associated (cas) genes utilize small RNAs for homology-directed detection and subsequent degradation of invading genetic elements, and they have been classified into three main types (I-III). Type III complexes share the Cas10 subunit but are subclassifed as type IIIA (CSM) and type IIIB (CMR), depending on their specificity for DNA or RNA targets, respectively. The role of CSM in limiting the spread of conjugative plasmids in Staphylococcus epidermidis was first described in 2008. Here, we report a detailed investigation of the composition and structure of the CSM complex from the archaeon Sulfolobus solfataricus, using a combination of electron microscopy, mass spectrometry, and deep sequencing. This reveals a three-dimensional model for the CSM complex that includes a helical component strikingly reminiscent of the backbone structure of the type I (Cascade) family.

Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity.

The prokaryotic clusters of regularly interspaced palindromic repeats (CRISPR) system utilizes genomically encoded CRISPR RNA (crRNA), derived from invading viruses and incorporated into ribonucleoprotein complexes with CRISPR-associated (CAS) proteins, to target and degrade viral DNA or RNA on subsequent infection. RNA is targeted by the CMR complex. In Sulfolobus solfataricus, this complex is composed of seven CAS protein subunits (Cmr1-7) and carries a diverse "payload" of targeting crRNA. The crystal structure of Cmr7 and low-resolution structure of the complex are presented. S. solfataricus CMR cleaves RNA targets in an endonucleolytic reaction at UA dinucleotides. This activity is dependent on the 8 nt repeat-derived 5' sequence in the crRNA, but not on the presence of a protospacer-associated motif (PAM) in the target. Both target and guide RNAs can be cleaved, although a single molecule of guide RNA can support the degradation of multiple targets.

Antimicrobial and host cell-directed activities of Gly/Ser-rich peptides from salmonid cathelicidins.

Cathelicidins, a major family of vertebrate antimicrobial peptides (AMPs), have a recognized role in the first line of defense against infections. They have been identified in several salmonid species, where the putative mature peptides are unusually long and rich in serine and glycine residues, often arranged in short multiple repeats (RLGGGS/RPGGGS) intercalated by hydrophobic motifs. Fragments of 24-40 residues, spanning specific motifs and conserved sequences in grayling or brown, rainbow and brook trout, were chemically synthesized and examined for antimicrobial activity against relevant Gram-positive and Gram-negative salmonid pathogens, as well as laboratory reference strains. They were not active in complete medium, but showed varying potency and activity spectra in diluted media. Bacterial membrane permeabilization also occurred only under these conditions and was indicated by rapid propidium iodide uptake in peptide-treated bacteria. However, circular dichroism analyses indicated that they did not significantly adopt ordered conformations in membrane-like environments. The peptides were not hemolytic or cytotoxic to trout cells, including freshly purified head kidney leukocytes (HKL) and the fibroblastic RTG-2 cell line. Notably, when exposed to them, HKL showed increased metabolic activity, while a growth-promoting effect was observed on RTG-2 cells, suggesting a functional interaction of salmonid cathelicidins with host cells similar to that shown by mammalian ones. The three most active peptides produced a dose-dependent increase in phagocytic uptake by HKL simultaneously stimulated with bacterial particles. The peptide STF(1-37), selected for further analyses, also enhanced phagocytic uptake in the presence of autologous serum, and increased intracellular killing of live E. coli. Furthermore, when tested on HKL in combination with the immunostimulant ß-glucan, it synergistically potentiated both phagocytic uptake and the respiratory burst response, activities that play a key role in fish immunity. Collectively, these data point to a role of salmonid cathelicidins as modulators of fish microbicidal mechanisms beyond a salt-sensitive antimicrobial activity, and encourage further studies also in view of potential applications in aquaculture.

The architecture of an Okazaki fragment-processing holoenzyme from the archaeon Sulfolobus solfataricus.

DNA replication on the lagging strand occurs via the synthesis and maturation of Okazaki fragments. In archaea and eukaryotes, the enzymatic activities required for this process are supplied by a replicative DNA polymerase, Flap endonuclease 1 (Fen1) and DNA ligase 1 (Lig1). These factors interact with the sliding clamp PCNA (proliferating cell nuclear antigen) providing a potential means of co-ordinating their sequential actions within a higher order assembly. In hyperthermophilic archaea of the Sulfolobus genus, PCNA is a defined heterotrimeric assembly and each subunit interacts preferentially with specific client proteins. We have exploited this inherent asymmetry to assemble a PCNA-polymerase-Fen1-ligase complex on DNA and have visualized it by electron microscopy. Our studies reveal the structural basis of co-occupancy of a single PCNA ring by the three distinct client proteins.

Electron microscopy studies of Type III CRISPR machines in Sulfolobus solfataricus.

The CRISPR (clustered regularly interspaced short palindromic repeats) system is an adaptive immune system that targets viruses and other mobile genetic elements in bacteria and archaea. Cells store information of past infections in their genome in repeat-spacer arrays. After transcription, these arrays are processed into unit-length crRNA (CRISPR RNA) that is loaded into effector complexes encoded by Cas (CRISPR-associated) genes. CRISPR-Cas complexes target invading nucleic acid for degradation. CRISPR effector complexes have been classified into three main types (I-III). Type III effector complexes share the Cas10 subunit. In the present paper, we discuss the structures of the two Type III effector complexes from Sulfolobus solfataricus, SsoCSM (subtype III-A) and SsoCMR (subtype III-B), obtained by electron microscopy and single particle analysis. We also compare these structures with Cascade (CRISPR-associated complex for antiviral defence) and with the RecA nucleoprotein.

Structure of the Saccharolobus solfataricus type III-D CRISPR effector.

CRISPR-Cas is a prokaryotic adaptive immune system, classified into six different types, each characterised by a signature protein. Type III systems, classified based on the presence of a Cas10 subunit, are rather diverse multi-subunit assemblies with a range of enzymatic activities and downstream ancillary effectors. The broad array of current biotechnological CRISPR applications is mainly based on proteins classified as Type II, however recent developments established the feasibility and efficacy of multi-protein Type III CRISPR-Cas effector complexes as RNA-targeting tools in eukaryotes. The crenarchaeon Saccharolobus solfataricus has two type III system subtypes (III-B and III-D). Here, we report the cryo-EM structure of the Csm Type III-D complex from S. solfataricus (SsoCsm), which uses CRISPR RNA to bind target RNA molecules, activating the Cas10 subunit for antiviral defence. The structure reveals the complex organisation, subunit/subunit connectivity and protein/guide RNA interactions of the SsoCsm complex, one of the largest CRISPR effectors known.

[Recent advances in the structural biology of the class C G protein-coupled receptors: The metabotropic Glutamate receptor 5]. / Les avancées récentes dans le domaine de la biologie structurale des récepteurs couplés aux protéines G de la classe C : Le récepteur métabotropique du glutamate 5.

Class C GPCRs, that include metabotropic glutamate receptors (mGlu), taste receptors, GABAB receptor and Calcium-sensing receptor, are unusual in terms of their molecular architecture and allosteric regulation. They all form obligatory dimers, dimerization being fundamental for their function. More specifically, the mGlu are activated by the main excitatory neurotransmitter, L-glutamate. mGlu activation by glutamate binding in the venus flytrap domain (VFT) triggers conformational changes that are transmitted, through the Cystein-Rich Domain (CRD), to the conserved fold of 7 transmembrane helices (7TM), that couples to intracellular G protein. mGlu activity can also be allosterically modulated by positive (PAM) or negative (NAM) allosteric modulators binding to the 7TM. Recent progress in cryo-electron microscopy (cryoEM) has allowed unprecedented advances in deciphering the structural and molecular basis of their activation mechanism. The agonist induces a large movement between the subunits, bringing the 7TMs together and stabilizing a 7TM conformation structurally similar to the inactive state. The diversity of inactive conformations for the class C was unexpected but allows PAM stabilising a 7TM active conformation independent of the conformational changes induced by agonists, representing an alternative mode of mGlu activation. Here we present and discuss recent structural characterisation of mGlu receptors, highlighting findings that make the class C of GPCR unique. Understanding the structural basis of mGlu dimer signaling represents a landmark achievement and paves the way for structural investigation of GPCR dimer signaling in general. Structural information will open new avenues for structure-based drug design.

Title: Les avancées récentes dans le domaine de la biologie structurale des récepteurs couplés aux protéines G de la classe C : Le récepteur métabotropique du glutamate 5. Abstract: La classe C des Récepteurs Couplés aux Protéines G (RCPG) comprend plusieurs membres aux fonctions physiologiques importantes comme par exemple les récepteurs des principaux neurotransmetteurs excitateurs (glutamate) et inhibiteurs (GABA) du système nerveux, les récepteurs des goûts umami et sucré et les récepteurs sensibles au calcium. Ces récepteurs possèdent une architecture moléculaire particulière, caractérisée par la présence d'un large domaine extracellulaire (ECD) relié à un domaine membranaire composé de 7 hélices transmembranaires (7TM). De plus, ils forment tous des dimères obligatoires, la dimérisation étant fondamentale pour leur fonction. La fixation d'agoniste dans l'ECD induit l'activation du récepteur. L'activité des agonistes peut être modulée de manière allostérique par des modulateurs positifs (PAM) ou négatifs (NAM), se liant au domaine 7TM. Il est important de comprendre comment les changements de conformation induits par la liaison des agonistes au sein du domaine extracellulaire sont transmis au domaine transmembranaire mais aussi de comprendre les bases structurales et moléculaires de la régulation allostérique des récepteurs de la classe C. Les progrès récents de la microscopie électronique en conditions cryogéniques (cryoEM) ont permis des avancées sans précédent dans le décryptage des bases structurelles et moléculaires des mécanismes d'activation des RCPG de classe C, et notamment du récepteur métabotropique du glutamate de type 5 (mGlu5). Le glutamate entraîne une fermeture et un changement d'orientation des domaines extracellulaires qui induit un mouvement important entre les sous-unités, rapprochant les 7TM et stabilisant la conformation active du récepteur. La diversité de conformations inactives pour les récepteurs de la classe C était inattendue mais propice à une activation possible par des PAM. Ces derniers stabilisent une conformation active des 7TM, indépendante des changements conformationnels induits par les agonistes, représentant un mode alternatif d'activation des récepteurs mGlu. Nous présentons et discutons ici les caractérisations structurales récentes des récepteurs de classe C, en soulignant les résultats qui rendent cette famille de récepteurs unique. La compréhension de la base structurelle de la signalisation des dimères de mGlu représente une réalisation historique et ouvre la voie à l'analyse de la signalisation des dimères de RCPG en général. Ces analyses structurales devraient également ouvrir de nouvelles voies pour la conception de médicaments ciblant cette famille de récepteurs qui sont aussi des cibles thérapeutiques.

Agonists and allosteric modulators promote signaling from different metabotropic glutamate receptor 5 conformations.

Metabotropic glutamate receptors (mGluRs) are dimeric G-protein-coupled receptors activated by the main excitatory neurotransmitter, L-glutamate. mGluR activation by agonists binding in the venus flytrap domain is regulated by positive (PAM) or negative (NAM) allosteric modulators binding to the 7-transmembrane domain (7TM). We report the cryo-electron microscopy structures of fully inactive and intermediate-active conformations of mGlu5 receptor bound to an antagonist and a NAM or an agonist and a PAM, respectively, as well as the crystal structure of the 7TM bound to a photoswitchable NAM. The agonist induces a large movement between the subunits, bringing the 7TMs together and stabilizing a 7TM conformation structurally similar to the inactive state. Using functional approaches, we demonstrate that the PAM stabilizes a 7TM active conformation independent of the conformational changes induced by agonists, representing an alternative mode of mGlu activation. These findings provide a structural basis for different mGluR activation modes.

Molecular basis for metabolite channeling in a ring opening enzyme of the phenylacetate degradation pathway.

Substrate channeling is a mechanism for the internal transfer of hydrophobic, unstable or toxic intermediates from the active site of one enzyme to another. Such transfer has previously been described to be mediated by a hydrophobic tunnel, the use of electrostatic highways or pivoting and by conformational changes. The enzyme PaaZ is used by many bacteria to degrade environmental pollutants. PaaZ is a bifunctional enzyme that catalyzes the ring opening of oxepin-CoA and converts it to 3-oxo-5,6-dehydrosuberyl-CoA. Here we report the structures of PaaZ determined by electron cryomicroscopy with and without bound ligands. The structures reveal that three domain-swapped dimers of the enzyme form a trilobed structure. A combination of small-angle X-ray scattering (SAXS), computational studies, mutagenesis and microbial growth experiments suggests that the key intermediate is transferred from one active site to the other by a mechanism of electrostatic pivoting of the CoA moiety, mediated by a set of conserved positively charged residues.

The structure of bactofilin filaments reveals their mode of membrane binding and lack of polarity.

Bactofilins are small ß-helical proteins that form cytoskeletal filaments in a range of bacteria. Bactofilins have diverse functions, from cell stalk formation in Caulobacter crescentus to chromosome segregation and motility in Myxococcus xanthus. However, the precise molecular architecture of bactofilin filaments has remained unclear. Here, sequence analysis and electron microscopy results reveal that, in addition to being widely distributed across bacteria and archaea, bactofilins are also present in a few eukaryotic lineages such as the Oomycetes. Electron cryomicroscopy analysis demonstrated that the sole bactofilin from Thermus thermophilus (TtBac) forms constitutive filaments that polymerize through end-to-end association of the ß-helical domains. Using a nanobody, we determined the near-atomic filament structure, showing that the filaments are non-polar. A polymerization-impairing mutation enabled crystallization and structure determination, while reaffirming the lack of polarity and the strength of the ß-stacking interface. To confirm the generality of the lack of polarity, we performed coevolutionary analysis on a large set of sequences. Finally, we determined that the widely conserved N-terminal disordered tail of TtBac is responsible for direct binding to lipid membranes, both on liposomes and in Escherichia coli cells. Membrane binding is probably a common feature of these widespread but only recently discovered filaments of the prokaryotic cytoskeleton.

Structure of an octameric form of the minichromosome maintenance protein from the archaeon Pyrococcus abyssi.

Cell division is a complex process that requires precise duplication of genetic material. Duplication is concerted by replisomes. The Minichromosome Maintenance (MCM) replicative helicase is a crucial component of replisomes. Eukaryotic and archaeal MCM proteins are highly conserved. In fact, archaeal MCMs are powerful tools for elucidating essential features of MCM function. However, while eukaryotic MCM2-7 is a heterocomplex made of different polypeptide chains, the MCM complexes of many Archaea form homohexamers from a single gene product. Moreover, some archaeal MCMs are polymorphic, and both hexameric and heptameric architectures have been reported for the same polypeptide. Here, we present the structure of the archaeal MCM helicase from Pyrococcus abyssi in its single octameric ring assembly. To our knowledge, this is the first report of a full-length octameric MCM helicase.

Crystal structures of malonyl-coenzyme A decarboxylase provide insights into its catalytic mechanism and disease-causing mutations.

Malonyl-coenzyme A decarboxylase (MCD) is found from bacteria to humans, has important roles in regulating fatty acid metabolism and food intake, and is an attractive target for drug discovery. We report here four crystal structures of MCD from human, Rhodopseudomonas palustris, Agrobacterium vitis, and Cupriavidus metallidurans at up to 2.3 Å resolution. The MCD monomer contains an N-terminal helical domain involved in oligomerization and a C-terminal catalytic domain. The four structures exhibit substantial differences in the organization of the helical domains and, consequently, the oligomeric states and intersubunit interfaces. Unexpectedly, the MCD catalytic domain is structurally homologous to those of the GCN5-related N-acetyltransferase superfamily, especially the curacin A polyketide synthase catalytic module, with a conserved His-Ser/Thr dyad important for catalysis. Our structures, along with mutagenesis and kinetic studies, provide a molecular basis for understanding pathogenic mutations and catalysis, as well as a template for structure-based drug design.