8 Dec 2020

In situ structural analysis of SARS-CoV-2 spike reveals flexibility mediated by three hinges (Science)

The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required for cell entry and is the primary focus for vaccine development. In this study, G. Hummer, J.K. Locker, M. Beck et.al. combined cryo–electron tomography, subtomogram averaging, and molecular dynamics simulations to structurally analyze S in situ. Compared with the recombinant S, the viral S was more heavily glycosylated and occurred mostly in the closed prefusion conformation. They show that the stalk domain of S contains three hinges, giving the head unexpected orientational freedom. They propose that the hinges allow S to scan the host cell surface, shielded from antibodies by an extensive glycan coat. The structure of native S contributes to our understanding of SARS-CoV-2 infection and potentially to the development of safe vaccines.

8 Dec 2020

Mapping Neutralizing and Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain by Structure-Guided High-Resolution Serology (Cell)

Analysis of the specificity and kinetics of neutralizing antibodies (nAbs) elicited by SARS-CoV-2 infection is crucial for understanding immune protection and identifying targets for vaccine design. In a cohort of 647 SARS-CoV-2-infected subjects, D. Corti, D. Veesler et. al. found that both the magnitude of Ab responses to SARS-CoV-2 spike (S) and nucleoprotein and nAb titers correlate with clinical scores. The receptor-binding domain (RBD) is immunodominant and the target of 90% of the neutralizing activity present in SARS-CoV-2 immune sera. Whereas overall RBD-specific serum IgG titers waned with a half-life of 49 days, nAb titers and avidity increased over time for some individuals, consistent with affinity maturation. They structurally defined an RBD antigenic map and serologically quantified serum Abs specific for distinct RBD epitopes leading to the identification of two major receptor-binding motif antigenic sites. Their results explain the immunodominance of the receptor-binding motif and will guide the design of COVID-19 vaccines and therapeutics.

11 Nov 2020

Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes (Cell Reports)

Biotin-labeled molecular probes, comprising specific regions of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. Here, Shapiro, L., Kwong, P.D. et. al. designed constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions include full-length spike ectodomain as well as various subregions, and we also design mutants that eliminate recognition of the angiotensin-converting enzyme 2 (ACE2) receptor. Yields of biotin-labeled probes from transient transfection range from similar to 0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes are characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe is determined by cryoelectron microscopy. They also characterize antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike ectodomain probes.

18 Dec 2020

Structural and dynamics analysis of intrinsically disordered proteins by high-speed atomic force microscopy (Nature Nanotechnology)

Intrinsically disordered proteins (IDPs) are ubiquitous proteins that are disordered entirely or partly and play important roles in diverse biologicalphenomena. Their structure dynamically samples a multitude of conformational states, thus rendering their structural analysis very difficult. Here S. Longhi, T. Ando. Et al. explore the potential of high-speed atomic force microscopy (HS-AFM) for characterizing the structure and dynamics of IDPs. Successive HS-AFM images of an IDP molecule can not only identify constantly folded and constantly disordered regions in the molecule, but can also document disorder-to-order transitions. Moreover, the number of amino acids contained in these disordered regions can be roughly estimated, enabling a semiquantitative, realistic description of the dynamic structure of IDPs.

18 Dec 2020

Small-molecule-induced polymerization triggers degradation of BCL6 (Nature)

Effective and sustained inhibition of non-enzymatic oncogenic driver proteins is a major pharmacological challenge. The clinical success of thalidomide analogues demonstrates the therapeutic efficacy of drug-induced degradation of transcription factors and other cancer targets(1-3), but a substantial subset of proteins are resistant to targeted degradation using existing approaches(4,5). B. Elber, S. Fischer et.al. report an alternative mechanism of targeted protein degradation, in which a small molecule induces the highly specific, reversible polymerization of a target protein, followed by its sequestration into cellular foci and subsequent degradation. BI-3802 is a small molecule that binds to the Broad-complex, Tramtrack and Bric-a-brac (BTB) domain of the oncogenic transcription factor B cell lymphoma 6 (BCL6) and leads to the proteasomal degradation of BCL6(6). They use cryo-electron microscopy to reveal how the solvent-exposed moiety of a BCL6-binding molecule contributes to a composite ligand-protein surface that engages BCL6 homodimers to form a supramolecular structure. Drug-induced formation of BCL6 filaments facilitates ubiquitination by the SIAH1 E3 ubiquitin ligase. Our findings demonstrate that a small molecule such as BI-3802 can induce polymerization coupled to highly specific protein degradation, which in the case of BCL6 leads to increased pharmacological activity compared to the effects induced by other BCL6 inhibitors. These findings open new avenues for the development of therapeutic agents and synthetic biology.

8 Dec 2020

Visualizing a protonated RNA state that modulates microRNA-21 maturation (Nature Chemical Biology)

MicroRNAs are evolutionarily conserved small, noncoding RNAs that regulate diverse biological processes. Due to their essential regulatory roles, microRNA biogenesis is tightly regulated, where protein factors are often found to interact with specific primary and precursor microRNAs for regulation. Here, using NMR relaxation dispersion spectroscopy and mutagenesis, Qi Zhang et. al. reveal that the precursor of oncogenic microRNA-21 exists as a pH-dependent ensemble that spontaneously reshuffles the secondary structure of the entire apical stem-loop region, including the Dicer cleavage site. They show that the alternative excited conformation transiently sequesters the bulged adenine into a noncanonical protonated A⁺-G mismatch, conferring a substantial enhancement in Dicer processing over its ground conformational state. These results indicate that microRNA maturation efficiency may be encoded in the intrinsic dynamic ensemble of primary and precursor microRNAs, providing a potential means of regulating microRNA biogenesis in response to environmental and cellular stimuli.

8 Dec 2020

Three-dimensional deconvolution processing for STEM cryotomography (PNAS)

The complex environment of biological cells and tissues has motivated development of three-dimensional (3D) imaging in both light and electron microscopies. To this end, one of the primary tools in fluorescence microscopy is that of computational deconvolution. Wide-field fluorescence images are often corrupted by haze due to out-of-focus light, i.e., to cross-talk between different object planes as represented in the 3D image. Using prior understanding of the image formation mechanism, it is possible to suppress the cross-talk and reassign the unfocused light to its proper source post facto. Electron tomography based on tilted projections also exhibits a cross-talk between distant planes due to the discrete angular sampling and limited tilt range. By use of a suitably synthesized 3D point spread function, M. Elbaum, J.W. Sedat et.al.show here that deconvolution leads to similar improvements in volume data reconstructed from cryoscanning transmission electron tomography (CSTET), namely a dramatic in-plane noise reduction and improved representation of features in the axial dimension. Contrast enhancement is demonstrated first with colloidal gold particles and then in representative cryotomograms of intact cells. Deconvolution of CSTET data collected from the periphery of an intact nucleus revealed partially condensed, extended structures in interphase chromatin.

8 Dec 2020

The Active Site of a Prototypical "Rigid" Drug Target is Marked by Extensive Conformational Dynamics (Angewandte Chemie Int. Ed.)

Drug discovery, in particular optimization of candidates using medicinal chemistry, is generally guided by structural biology. However, for optimizing binding kinetics, relevant for efficacy and off-target effects, information on protein motion is important. Herein, R. Linser et. al. demonstrate for the prototypical textbook example of an allegedly "rigid protein" that substantial active-site dynamics have generally remained unrecognized, despite thousands of medicinal-chemistry studies on this model over decades. Comparing cryogenic X-ray structures, solid-state NMR on micro-crystalline protein at room temperature, and solution NMR structure and dynamics, supported by MD simulations, they show that under physiologically relevant conditions the pocket is in fact shaped by pronounced open/close conformational-exchange dynamics. The study, which is of general significance for pharmacological research, evinces a generic pitfall in drug discovery routines.

11 Nov 2020

Structure of inhibitor-bound mammalian complex I (Nat. Commun.)

Respiratory complex I (NADH:ubiquinone oxidoreductase) captures the free energy from oxidising NADH and reducing ubiquinone to drive protons across the mitochondrial inner membrane and power oxidative phosphorylation. Recent cryo-EM analyses have produced near-complete models of the mammalian complex but leave the molecular principles of its long-range energy coupling mechanism open to debate. Here, V.R.I. Kaila, J. Hirst et. al.  describe the 3.0-Ao resolution cryo-EM structure of complex I from mouse heart mitochondria with a substrate-like inhibitor, piericidin A, bound in the ubiquinone-binding active site. They combine the structural analyses with both functional and computational studies to demonstrate competitive inhibitor binding poses and provide evidence that two inhibitor molecules bind end-to-end in the long substrate binding channel. Their findings reveal information about the mechanisms of inhibition and substrate reduction that are central for understanding the principles of energy transduction in mammalian complex I. The respiratory complex I (NADH:ubiquinone oxidoreductase) is a large redox-driven proton pump that initiates respiration in mitochondria. Here, the authors present the 3.0 angstrom cryo-EM structure of complex I from mouse heart mitochondria with the ubiquinone-analogue inhibitor piericidin A bound in the active site and with kinetic measurements and MD simulations they further show that this inhibitor acts competitively against the native ubiquinone-10 substrate.

11 Nov 2020

Analysis of protein-DNA interactions in chromatin by UV induced cross-linking and mass spectrometry (Nat. Commun.)

Protein-DNA interactions are key to the functionality and stability of the genome. Identification and mapping of protein-DNA interaction interfaces and sites is crucial for understanding DNA-dependent processes. Here, H. Urlaub et. al. present a workflow that allows mass spectrometric (MS) identification of proteins in direct contact with DNA in reconstituted and native chromatin after cross-linking by ultraviolet (UV) light. Their approach enables the determination of contact interfaces at amino-acid level. With the example of chromatin-associated protein SCML2 they show that the technique allows differentiation of nucleosome-binding interfaces in distinct states. By UV cross-linking of isolated nuclei, they determined the cross-linking sites of several factors including chromatin-modifying enzymes, demonstrating that their workflow is not restricted to reconstituted materials. As the approach can distinguish between protein-RNA and DNA interactions in one single experiment, they project that it will be possible to obtain insights into chromatin and its regulation in the future. Cross-linking mass spectrometry (XLMS) allows mapping of protein-protein and protein-RNA interactions, but the analysis of protein-DNA complexes remains challenging. Here, the authors develop a UV light-based XLMS workflow to determine protein-DNA interfaces in reconstituted chromatin and isolated nuclei.

2 Nov 2020

Sensitivity enhancement of homonuclear multidimensional NMR correlations for labile sites in proteins, polysaccharides, and nucleic acids (Nat. Commun.)

Multidimensional TOCSY and NOESY are central experiments in chemical and biophysical NMR. Limited efficiencies are an intrinsic downside of these methods, particularly when targeting labile sites. This study reported by L. Frydman et. al. demonstrates that the decoherence imparted on these protons through solvent exchanges can, when suitably manipulated, lead to dramatic sensitivity gains per unit time in the acquisition of these experiments. To achieve this, a priori selected frequencies are encoded according to Hadamard recipes, while concurrently subject to looped selective inversion or selective saturation procedures. Suitable processing then leads to protein, oligosaccharide and nucleic acid cross-peak enhancements of ≈200–1000% per scan, in measurements that are ≈10-fold faster than conventional counterparts. The extent of these gains will depend on the solvent exchange and relaxation rates of the targeted sites; these gains also benefit considerably from the spectral resolution provided by ultrahigh fields, as corroborated by NMR experiments at 600 MHz and 1 GHz. The mechanisms underlying these experiments’ enhanced efficiencies are analyzed on the basis of three-way polarization transfer interplays between the water, labile and non-labile protons, and the experimental results are rationalized using both analytical and numerical derivations. Limitations as well as further extensions of the proposed methods, are also discussed.