17 Aug 2021

Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome (Science)

Programmed ribosomal frameshifting is a key event during translation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA genome that allows synthesis of the viral RNA-dependent RNA polymerase and downstream proteins. Here, N. Ban, J.F. Atkinson et al. present the cryo–electron microscopy structure of a translating mammalian ribosome primed for frameshifting on the viral RNA. The viral RNA adopts a pseudoknot structure that lodges at the entry to the ribosomal messenger RNA (mRNA) channel to generate tension in the mRNA and promote frameshifting, whereas the nascent viral polyprotein forms distinct interactions with the ribosomal tunnel.Biochemical experiments validate the structural observations and reveal mechanistic and regulatory features that influence frameshifting efficiency. Finally, they compare compounds previously shown to reduce frameshifting with respect to their ability to inhibit SARS-CoV-2 replication, establishing coronavirus frameshifting as a target for antiviral intervention.

4 Aug 2021

Structural basis for SARS-CoV-2 envelope protein recognition of human cell junction protein PALS1 (Nature Communications)

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has created global health and economic emergencies. SARS-CoV-2 viruses promote their own spread and virulence by hijacking human proteins, which occurs through viral protein recognition of human targets. To understand the structural basis for SARS-CoV-2 viral-host protein recognition, here we use cryo-electron microscopy (cryo-EM) to determine a complex structure of the human cell junction protein PALS1 and SARS-CoV-2 viral envelope (E) protein. Our reported structure shows that the E protein C-terminal DLLV motif recognizes a pocket formed exclusively by hydrophobic residues from the PDZ and SH3 domains of PALS1. Our structural analysis provides an explanation for the observation that the viral E protein recruits PALS1 from lung epithelial cell junctions. In addition, our structure provides novel targets for peptide- and small-molecule inhibitors that could block the PALS1-E interactions to reduce E-mediated virulence. SARS-CoV-2 viruses are known to hijack human proteins in order to facilitate their own virulence and replication. In this study, Liu and colleagues present structural analysis of how this phenomenon occurs between SARS-CoV-2 viral envelope protein and human PALS1. The findings provide insights in to viral-host recognition.

4 Aug 2021

An infectivity-enhancing site on the SARS-CoV-2 spike protein targeted by antibodies (Cell)

Antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein prevent SARSCoV-2 infection. However, the effects of antibodies against other spike protein domains are largely unknown. Here, we screened a series of anti-spike monoclonal antibodies from coronavirus disease 2019 (COVID-19) patients and found that some of antibodies against the N-terminal domain (NTD) induced the open conformation of RBD and thus enhanced the binding capacity of the spike protein to ACE2 and infectivity of SARSCoV-2. Mutational analysis revealed that all of the infectivity-enhancing antibodies recognized a specific site on the NTD. Structural analysis demonstrated that all infectivity-enhancing antibodies bound to NTD in a similar manner. The antibodies against this infectivity-enhancing site were detected at high levels in severe patients. Moreover, we identified antibodies against the infectivity-enhancing site in uninfected donors, albeit at a lower frequency. These findings demonstrate that not only neutralizing antibodies but also enhancing antibodies are produced during SARS-CoV-2 infection.

6 Oct 2022

Molecular architecture of the augmin complex (Nature Communications)

Accurate segregation of chromosomes during mitosis depends on the correct assembly of the mitotic spindle, a bipolar structure composed mainly of microtubules. The augmin complex, or homologous to augmin subunits (HAUS) complex, is an eight-subunit protein complex required for building robust mitotic spindles in metazoa. Augmin increases microtubule density within the spindle by recruiting the γ-tubulin ring complex (γ-TuRC) to pre-existing microtubules and nucleating branching microtubules. Here, we elucidate the molecular architecture of augmin by single particle cryo-electron microscopy (cryo-EM), computational methods, and crosslinking mass spectrometry (CLMS). Augmin’s highly flexible structure contains a V-shaped head and a filamentous tail, with the head existing in either extended or contracted conformational states. Our work highlights how cryo-EM, complemented by computational advances and CLMS, can elucidate the structure of a challenging protein complex and provides insights into the function of augmin in mediating microtubule branching nucleation.

6 Oct 2022

Plant receptor-like protein activation by a microbial glycoside hydrolase (Nature)

Plants rely on cell-surface-localized pattern recognition receptors to detect pathogen- or host-derived danger signals and trigger an immune response. Receptor-like proteins (RLPs) with a leucine-rich repeat (LRR) ectodomain constitute a subgroup of pattern recognition receptors and play a critical role in plant immunity. Mechanisms underlying ligand recognition and activation of LRR-RLPs remain elusive. Here we report a crystal structure of the LRR-RLP RXEG1 from Nicotiana benthamiana that recognizes XEG1 xyloglucanase from the pathogen Phytophthora sojae. The structure reveals that specific XEG1 recognition is predominantly mediated by an amino-terminal and a carboxy-terminal loop-out region (RXEG1(ID)) of RXEG1. The two loops bind to the active-site groove of XEG1, inhibiting its enzymatic activity and suppressing Phytophthora infection of N. benthamiana. Binding of XEG1 promotes association of RXEG1(LRR) with the LRR-type co-receptor BAK1 through RXEG1(ID) and the last four conserved LRRs to trigger RXEG1-mediated immune responses. Comparison of the structures of apo-RXEG1(LRR), XEG1–RXEG1(LRR) and XEG1–BAK1–RXEG1(LRR) shows that binding of XEG1 induces conformational changes in the N-terminal region of RXEG1(ID) and enhances structural flexibility of the BAK1-associating regions of RXEG1(LRR). These changes allow fold switching of RXEG1(ID) for recruitment of BAK1(LRR). Our data reveal a conserved mechanism of ligand-induced heterodimerization of an LRR-RLP with BAK1 and suggest a dual function for the LRR-RLP in plant immunity.

6 Oct 2022

In situ architecture of the ciliary base reveals the stepwise assembly of intraflagellar transport trains (Science)

The cilium is an antenna-like organelle that performs numerous cellular functions, including motility, sensing, and signaling.The base of the cilium contains a selective barrier that regulates the entry of large intraflagellar transport (IFT) trains, which carry cargo proteins required for ciliary assembly and maintenance. However, the native architecture of the ciliary base and the process of IFT train assembly remain unresolved. In this work, we used in situ cryo-electron tomography to reveal native structures of the transition zone region and assembling IFT trains at the ciliary base in Chlamydomonas. We combined this direct cellular visualization with ultrastructure expansion microscopy to describe the front-to-back stepwise assembly of IFT trains: IFT-B forms the backbone, onto which bind IFT-A, dynein-1b, and finally kinesin-2 before entry into the cilium.

6 Oct 2022

Molecular basis for the selective G protein signaling of somatostatin receptors (Nature Chemical Biology)

G protein-coupled receptors (GPCRs) modulate every aspect of physiological functions mainly through activating heterotrimeric G proteins. A majority of GPCRs promiscuously couple to multiple G protein subtypes. Here we validate that in addition to the well-known G_i/o pathway, somatostatin receptor 2 and 5 (SSTR2 and SSTR5) couple to the G_q/11 pathway and show that smaller ligands preferentially activate the G_i/o pathway. We further determined cryo-electron microscopy structures of the SSTR2‒G_o and SSTR2‒G_q complexes bound to octreotide and SST-14. Structural and functional analysis revealed that G protein selectivity of SSTRs is not only determined by structural elements in the receptor–G protein interface, but also by the conformation of the agonist-binding pocket. Accordingly, smaller ligands fail to stabilize a broader agonist-binding pocket of SSTRs that is required for efficient G_q/11coupling but not G_i/o coupling. Our studies facilitate the design of drugs with selective G protein signaling to improve therapeutic efficacy.

6 Oct 2022

Columnar structure of human telomeric chromatin (Nature)

Telomeres, the ends of eukaryotic chromosomes, play pivotal parts in ageing and cancer and are targets of DNA damage and the DNA damage response. Little is known about the structure of telomeric chromatin at the molecular level. Here we used negative stain electron microscopy and single-molecule magnetic tweezers to characterize 3-kbp-long telomeric chromatin fibres. We also obtained the cryogenic electron microscopy structure of the condensed telomeric tetranucleosome and its dinucleosome unit. The structure displayed close stacking of nucleosomes with a columnar arrangement, and an unusually short nucleosome repeat length that comprised about 132 bp DNA wound in a continuous superhelix around histone octamers. This columnar structure is primarily stabilized by the H2A carboxy-terminal and histone amino-terminal tails in a synergistic manner. The columnar conformation results in exposure of the DNA helix, which may make it susceptible to both DNA damage and the DNA damage response. The conformation also exists in an alternative open state, in which one nucleosome is unstacked and flipped out, which exposes the acidic patch of the histone surface. The structural features revealed in this work suggest mechanisms by which protein factors involved in telomere maintenance can access telomeric chromatin in its compact form.

6 Oct 2022

Scientists are using AI to dream up revolutionary new proteins (Nature)

Huge advances in artificial intelligence mean researchers can design completely original molecules in seconds instead of months. In June, South Korean regulators authorized the first-ever medicine, a COVID-19 vaccine, to be made from a novel protein designed by humans. The vaccine is based on a spherical protein ‘nanoparticle’ that was created by researchers nearly a decade ago, through a labour-intensive trial-and error-process1.

Now, thanks to gargantuan advances in artificial intelligence (AI), a team led by David Baker, a biochemist at the University of Washington (UW) in Seattle, reports in Science^2,3 that it can design such molecules in seconds instead of months.

9 Sep 2022

Cross-validation of distance measurements in proteins by PELDOR/DEER and single-molecule FRET (Nature Communications)

Pulsed electron-electron double resonance spectroscopy (PELDOR/DEER) and single-molecule Forster resonance energy transfer spectroscopy (smFRET) are frequently used to determine conformational changes, structural heterogeneity, and inter probe distances in biological macromolecules. They provide qualitative information that facilitates mechanistic understanding of biochemical processes and quantitative data for structural modelling. To provide a comprehensive comparison of the accuracy of PELDOR/DEER and smFRET, we use a library of double cysteine variants of four proteins that undergo large-scale conformational changes upon ligand binding. With either method, we use established standard experimental protocols and data analysis routines to determine inter-probe distances in the presence and absence of ligands. The results are compared to distance predictions from structural models. Despite an overall satisfying and similar distance accuracy, some inconsistencies are identified, which we attribute to the use of cryoprotectants for PELDOR/DEER and label-protein interactions for smFRET. This large-scale cross-validation of PELDOR/DEER and smFRET highlights the strengths, weaknesses, and synergies of these two important and complementary tools in integrative structural biology.

Pulsed electron-electron double resonance spectroscopy (PELDOR/DEER) and single-molecule Forster resonance energy transfer spectroscopy (smFRET) are used to determine conformational changes and probe distances in biological macromolecules. Here the authors compare the methods on a large set of samples.

9 Sep 2022

Toward protein NMR at physiological concentrations by hyperpolarized water-Finding and mapping uncharted conformational spaces (Science Advances)

Nuclear magnetic resonance (NMR) spectroscopy is a key method for determining the structural dynamics of proteins in their native solution state. However, the low sensitivity of NMR typically necessitates nonphysiologically high sample concentrations, which often limit the relevance of the recorded data. We show how to use hyperpolarized water by dissolution dynamic nuclear polarization (DDNP) to acquire protein spectra at concentrations of 1 μM within seconds and with a high signal-to-noise ratio. The importance of approaching physiological concentrations is demonstrated for the vital MYC-associated factor X, which we show to switch conformations when diluted. While in vitro conditions lead to a population of the well-documented dimer, concentrations lowered by more than two orders of magnitude entail dimer dissociation and formation of a globularly folded monomer. We identified this structure by integrating DDNP with computational techniques to overcome the often-encountered constraint of DDNP of limited structural information provided by the typically detected one-dimensional spectra.