9 May 2022

Secondary structural ensembles of the SARS-CoV-2 RNA genome in infected cells (Nature Communications)

SARS-CoV-2 is a betacoronavirus with a single-stranded, positive-sense, 30-kilobase RNA genome responsible for the ongoing COVID-19 pandemic. Although population average structure models of the genome were recently reported, there is little experimental data on native structural ensembles, and most structures lack functional characterization. Here we report secondary structure heterogeneity of the entire SARS-CoV-2 genome in two lines of infected cells at single nucleotide resolution. Our results reveal alternative RNA conformations across the genome and at the critical frameshifting stimulation element (FSE) that are drastically different from prevailing population average models. Importantly, we find that this structural ensemble promotes frameshifting rates much higher than the canonical minimal FSE and similar to ribosome profiling studies. Our results highlight the value of studying RNA in its full length and cellular context. The genomic structures detailed here lay groundwork for coronavirus RNA biology and will guide the design of SARS-CoV-2 RNA-based therapeutics.

9 May 2022

Pandemic strategies with computational and structural biology against COVID-19: A retrospective (Computational and Structural Biotechnology Journal)

The emergence of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which is the etiologic agent of the coronavirus disease 2019 (COVID-19) pandemic, has dominated all aspects of life since 2020. Research studies on the virus and exploration of therapeutic and preventive strategies have been moving at rapid rates to control the pandemic. In the field of bioinformatics or computational and structural biology, recent research strategies have used multiple disciplines to compile large datasets to uncover statistical correlations and significance, visualize and model proteins, perform molecular dynamics simulations, and employ the help of artificial intelligence and machine learning to harness computational processing power to further the research on COVID-19, including drug screening, drug design, vaccine development, prognosis prediction, and outbreak prediction. These recent developments should help us better understand the viral disease and develop the much-needed therapies and strategies for the management of COVID-19.

9 May 2022

Fighting SARS-CoV-2 with structural biology methods (Nature Methods)

Comment in Nature Methods highlights the importance of structural biology in fight against SAR-Cov-2. High-resolution structural information is critical for rapid development of vaccines and therapeutics against emerging human pathogens. Structural biology methods have been at the forefront of research on SARS-CoV-2 since the beginning of the COVID-19 pandemic. These technologies will continue to be powerful tools to fend off future public health threats.

11 May 2022

CENP-N promotes the compaction of centromeric chromatin (Nature Structural & Molecular Biology)

The histone variant CENP-A is the epigenetic determinant for the centromere, where it is interspersed with canonical H3 to form a specialized chromatin structure that nucleates the kinetochore. How nucleosomes at the centromere arrange into higher order structures is unknown. Here the authors demonstrate that the human CENP-A-interacting protein CENP-N promotes the stacking of CENP-A-containing mononucleosomes and nucleosomal arrays through a previously undefined interaction between the α6 helix of CENP-N with the DNA of a neighboring nucleosome. They describe the cryo-EM structures and biophysical characterization of such CENP-N-mediated nucleosome stacks and nucleosomal arrays and demonstrate that this interaction is responsible for the formation of densely packed chromatin at the centromere in the cell. Their results provide first evidence that CENP-A, together with CENP-N, promotes specific chromatin higher order structure at the centromere.

11 May 2022

True-atomic-resolution insights into the structure and functional role of linear chains and low-barrier hydrogen bonds in proteins (Nature Structural & Molecular Biology)

Hydrogen bonds are fundamental to the structure and function of biological macromolecules and have been explored in detail. The chains of hydrogen bonds (CHBs) and low-barrier hydrogen bonds (LBHBs) were proposed to play essential roles in enzyme catalysis and proton transport. However, high-resolution structural data from CHBs and LBHBs is limited. The challenge is that their ‘visualization’ requires ultrahigh-resolution structures of the ground and functionally important intermediate states to identify proton translocation events and perform their structural assignment. Ttrue-atomic-resolution structures of the light-driven proton pump bacteriorhodopsin, a model in studies of proton transport, show that CHBs and LBHBs not only serve as proton pathways, but also are indispensable for long-range communications, signaling and proton storage in proteins. The complete picture of CHBs and LBHBs discloses their multifunctional roles in providing protein functions and presents a consistent picture of proton transport and storage resolving long-standing debates and controversies.

11 May 2022

What's next for AlphaFold and the AI protein-folding revolution (Nature)

DeepMind software that can predict the 3D shape of proteins is already changing biology. This News Feature published in Nature lists several examples of successful deployment of AlphaFold and RoseTTAFold software and comments their performance.

6 Apr 2022

Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase alpha-aminoacrylate intermediate (PNAS)

NMR-assisted crystallography—the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry—holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5′-phosphate–dependent enzymes that catalyze β-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate β-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid–base catalytic residue βLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate C^β and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.

6 Apr 2022

Structure and mechanism of a methyltransferase ribozyme (Nature Chemical Biology)

Known ribozymes in contemporary biology perform a limited range of chemical catalysis, but in vitro selection has generated species that catalyze a broader range of chemistry; yet, there have been few structural and mechanistic studies of selected ribozymes. A ribozyme has recently been selected that can catalyze a site-specific methyl transfer reaction. The authors have solved the crystal structure of this ribozyme at a resolution of 2.3 Å, showing how the RNA folds to generate a very specific binding site for the methyl donor substrate. The structure immediately suggests a catalytic mechanism involving a combination of proximity and orientation and nucleobase-mediated general acid catalysis. The mechanism is supported by the pH dependence of the rate of catalysis. A selected methyltransferase ribozyme can thus use a relatively sophisticated catalytic mechanism, broadening the range of known RNA-catalyzed chemistry.

6 Apr 2022

Structural basis for mismatch surveillance by CRISPR–Cas9 (Nature)

CRISPR–Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage, and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood. Although Cas9 variants with greater discrimination against mismatches have been designed, these suffer from substantially reduced rates of on-target DNA cleavage. Here the authors used kinetics-guided cryo-electron microscopy to determine the structure of Cas9 at different stages of mismatch cleavage. Theyobserved a distinct, linear conformation of the guide RNA–DNA duplex formed in the presence of mismatches, which prevents Cas9 activation. Although the canonical kinked guide RNA–DNA duplex conformation facilitates DNA cleavage, they observe that substrates that contain mismatches distal to the protospacer adjacent motif are stabilized by reorganization of a loop in the RuvC domain. Mutagenesis of mismatch-stabilizing residues reduces off-target DNA cleavage but maintains rapid on-target DNA cleavage. By targeting regions that are exclusively involved in mismatch tolerance, they provide a proof of concept for the design of next-generation high-fidelity Cas9 variants.

6 Apr 2022

Structural basis of human telomerase recruitment by TPP1-POT1 (Science)

Telomerase maintains genome stability by extending the 3′ telomeric repeats at eukaryotic chromosome ends, thereby counterbalancing progressive loss caused by incomplete genome replication. In mammals, telomerase recruitment to telomeres is mediated by TPP1, which assembles as a heterodimer with POT1. The authors report cryo–electron microscopy

structures of DNA-bound telomerase in complex with TPP1 and with TPP1-POT1 at 3.2- and 3.9-angstrom resolution, respectively. Their structures define interactions between telomerase and TPP1-POT1 that are crucial for telomerase recruitment to telomeres. The presence of TPP1-POT1 stabilizes the DNA, revealing an unexpected path by which DNA exits the telomerase active site and a DNA anchor site on telomerase that is important for telomerase processivity. Their findings rationalize extensive prior genetic and biochemical findings and provide a framework for future mechanistic work on telomerase regulation.

6 Apr 2022

Structural basis for activation and gating of IP3receptors (Nature Communications)

A pivotal component of the calcium (Ca²⁺) signaling toolbox in cells is the inositol 1,4,5-triphosphate (IP₃) receptor (IP₃R), which mediates Ca²⁺ release from the endoplasmic reticulum (ER), controlling cytoplasmic and organellar Ca²⁺ concentrations. IP₃Rs are co-activated by IP₃ and Ca²⁺, inhibited by Ca²⁺ at high concentrations, and potentiated by ATP. However, the underlying molecular mechanisms are unclear. Here Erkan Karakas et. al. report cryo-electron microscopy (cryo-EM) structures of human type-3 IP₃R obtained from a single dataset in multiple gating conformations: IP₃-ATP bound pre-active states with closed channels, IP₃-ATP-Ca²⁺ bound active state with an open channel, and IP₃-ATP-Ca²⁺ bound inactive state with a closed channel.The structures demonstrate how IP₃-induced conformational changes prime the receptor for activation by Ca²⁺, how Ca²⁺ binding leads to channel opening, and how ATP modulates the activity, providing insights into the long-sought questions regarding the molecular mechanism underpinning receptor activation and gating.