8 Feb 2021

A trimeric human angiotensin-converting enzyme 2 as an anti-SARS-CoV-2 agent (Nature Struct.& Mol. Biol)

Effective intervention strategies are urgently needed to control the COVID-19 pandemic. Human angiotensin-converting enzyme 2 (ACE2) is a membrane-bound carboxypeptidase that forms a dimer and serves as the cellular receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ACE2 is also a key negative regulator of the renin–angiotensin sys- tem that modulates vascular functions. B. Chen et al. report here the properties of a trimeric ACE2 ectodomain variant, engineered using a structure-based approach. The trimeric ACE2 variant has a binding affinity of ~60 pM for the spike protein of SARS-CoV-2 (compared with 77 nM for monomeric ACE2 and 12–22 nM for dimeric ACE2 constructs), and its peptidase activity and the abil- ity to block activation of angiotensin II receptor type 1 in the renin–angiotensin system are preserved. Moreover, the engineered ACE2 potently inhibits SARS-CoV-2 infection in cell culture. These results suggest that engineered, trimeric ACE2 may be a promising anti-SARS-CoV-2 agent for treating COVID-19.

8 Feb 2021

Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 revealed by cryo-EM (Science Advances)

The recent outbreaks of SARS-CoV-2 pose a global health emergency. The SARS-CoV-2 trimeric spike (S) glyco- protein interacts with the human ACE2 receptor to mediate viral entry into host cells. Z. Huang, Y. Cong et al. report the cryo-EM structures of a tightly closed SARS-CoV-2 S trimer with packed fusion peptide and an ACE2-bound S trimer at 2.7- and 3.8-Å resolution, respectively. Accompanying ACE2 binding to the up receptor-binding domain (RBD), the associated ACE2-RBD exhibits continuous swing motions. Notably, the SARS-CoV-2 S trimer appears much more sensitive to the ACE2 receptor than the SARS-CoV S trimer regarding receptor-triggered transformation from the closed prefusion state to the fusion-prone open state, potentially contributing to the superior infectivity of SARS-CoV-2. They defined the RBD T470-T478 loop and Y505 as viral determinants for specific recognition of SARS-CoV-2 RBD by ACE2. Their findings depict the mechanism of ACE2-induced S trimer conformational transitions from the ground prefusion state toward the postfusion state, facilitating development of anti–SARS-CoV-2 vaccines and therapeutics.

8 Feb 2021

Cryo-EM Structure of an Extended SARS-CoV-2 Replication and Transcription Complex Reveals an Intermediate State in Cap Synthesis (Cell)

Transcription of SARS-CoV-2 mRNA requires sequential reactions facilitated by the replication and transcrip- tion complex (RTC). Here, Z. Rao, Z. Lou et al. present a structural snapshot of SARS-CoV-2 RTC as it transitions toward cap structure synthesis. They determine the atomic cryo-EM structure of an extended RTC assembled by nsp7- nsp82-nsp12-nsp132-RNA and a single RNA-binding protein, nsp9. Nsp9 binds tightly to nsp12 (RdRp) NiRAN, allowing nsp9 N terminus inserting into the catalytic center of nsp12 NiRAN, which then inhibits activity. They also show that nsp12 NiRAN possesses guanylyltransferase activity, catalyzing the formation of cap core structure (GpppA). The orientation of nsp13 that anchors the 50 extension of template RNA shows a remarkable conformational shift, resulting in zinc finger 3 of its ZBD inserting into a minor groove of paired template-primer RNA. These results reason an intermediate state of RTC toward mRNA synthesis, pave a way to understand the RTC architecture, and provide a target for antiviral development.

12 Jan 2021

Structural Basis of WLS/Evi-Mediated Wnt Transport and Secretion (Cell)

Wnts are evolutionarily conserved ligands that signal at short range to regulate morphogenesis, cell fate, and stem cell renewal. The first and essential steps in Wnt secretion are their O-palmitoleation and subsequent loading onto the dedicated transporter Wntless/evenness interrupted (WLS/Evi). D.M. Wirshup, F. Marcia et.al.  report the 3.2A resolution cryogenic electron microscopy (cryo-EM) structure of palmitoleated human WNT8A in complex with WLS. This is accompanied by biochemical experiments to probe the physiological implications of the observed association. The WLS membrane domain has close structural homology to G protein-coupled receptors (GPCRs). A Wnt hairpin inserts into a conserved hydrophobic cavity in the GPCR-like domain, and the palmitoleate protrudes between two helices into the bilayer. A conformational switch of highly conserved residues on a separate Wnt hairpin might contribute to its transfer to receiving cells. This work provides molecular-level insights into a central mechanism in animal body plan development and stem cell biology.

18 Dec 2020

Nearest-neighbor NMR spectroscopy: categorizing spectral peaks by their adjacent nuclei (Nature Communications)

Methyl-NMR enables atomic-resolution studies of structure and dynamics of large proteins in solution. However, resonance assignment remains challenging. The problem is to combine existing structural informational with sparse distance restraints and search for the most compatible assignment among the permutations. Prior classification of peaks as either from isoleucine, leucine, or valine reduces the search space by many orders of magnitude. However, this is hindered by overlapped leucine and valine frequencies. In contrast, the nearest-neighbor nuclei, coupled to the methyl carbons, resonate in distinct frequency bands. Here, Coote, P.,  Arthanari, H. et. al. develop a framework to imprint additional information about passively coupled resonances onto the observed peaks. This depends on simultaneously orchestrating closely spaced bands of resonances along different magnetization trajectories, using principles from control theory. For methyl-NMR, the method is implemented as a modification to the standard fingerprint spectrum (the 2D-HMQC). The amino acid type is immediately apparent in the fingerprint spectrum. There is no additional relaxation loss or an increase in experimental time. The method is validated on biologically relevant proteins. The idea of generating new spectral information using passive, adjacent resonances is applicable to other contexts in NMR spectroscopy. The structure and dynamics of large proteins and complexes can be studied by methyl-NMR but resonance assignment is still challenging. Here, the authors present a NMR method that leverages optimal control pulse design to unambiguously distinguish between Leu and Val using a simple 2D HMQC experiment and they apply it to several proteins including Cas9, interleukin, and human translation initiation factor eIF4a.

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.