11 Nov 2020

De novo design of picomolar SARS-CoV-2 miniprotein inhibitors (Science)

Targeting the interaction between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and the human angiotensin-converting enzyme 2 (ACE2) receptor is a promising therapeutic strategy. D. Baker et.al. designed inhibitors using two de novo design approaches. Computer-generated scaffolds were either built around an ACE2 helix that interacts with the spike receptor binding domain (RBD) or docked against the RBD to identify new binding modes, and their amino acid sequences were designed to optimize target binding, folding, and stability. Ten designs bound the RBD, with affinities ranging from 100 picomolar to 10 nanomolar, and blocked SARS-CoV-2 infection of Vero E6 cells with median inhibitory concentration (IC50) values between 24 picomolar and 35 nanomolar. The most potent, with new binding modes, are 56- and 64-residue proteins (IC50 similar to 0.16 nanograms per milliliter). Cryo-electron microscopy structures of these minibinders in complex with the SARS-CoV-2 spike ectodomain trimer with all three RBDs bound are nearly identical to the computational models. These hyperstable minibinders provide starting points for SARS-CoV-2 therapeutics.

27 Oct 2020

Structure-guided covalent stabilization of coronavirus spike glycoprotein trimers in the closed conformation (Nat. Struct. Mol. Biol.)

SARS-CoV-2 is the causative agent of the COVID-19 pandemic, with 10 million infections and more than 500,000 fatalities by June 2020. To initiate infection, the SARS-CoV-2 spike (S) glycoprotein promotes attachment to the host cell surface and fusion of the viral and host membranes. Prefusion SARS-CoV-2 S is the main target of neutralizing antibodies and the focus of vaccine design. However, its limited stability and conformational dynamics are limiting factors for developing countermeasures against this virus. Veesler, D. et. al. report here the design of a construct corresponding to the prefusion SARS-CoV-2 S ectodomain trimer, covalently stabilized by a disulfide bond in the closed conformation. Structural and antigenicity analyses show that they successfully shut S in the closed state without otherwise altering its architecture. They demonstrate that this strategy is applicable to other beta-coronaviruses, such as SARS-CoV and MERS-CoV, and might become an important tool for structural biology, serology, vaccine design and immunology studies.

27 Oct 2020

A thermostable, closed SARS-CoV-2 spike protein trimer (Nat. Struct. Mol. Biol.)

The spike (S) protein of SARS-CoV-2 mediates receptor binding and cell entry and is the dominant target of the immune system. It exhibits substantial conformational flexibility. It transitions from closed to open conformations to expose its receptor-binding site and, subsequently, from prefusion to postfusion conformations to mediate fusion of viral and cellular membranes. S-protein derivatives are components of vaccine candidates and diagnostic assays, as well as tools for research into the biology and immunology of SARS-CoV-2. Here Xiong, X.L., Briggs, J.A.G. et. al. have designed mutations in S that allow the production of thermostable, disulfide-bonded S-protein trimers that are trapped in the closed, prefusion state. Structures of the disulfide-stabilized and non-disulfide-stabilized proteins reveal distinct closed and locked conformations of the S trimer. They demonstrate that the designed, thermostable, closed S trimer can be used in serological assays. This protein has potential applications as a reagent for serology, virology and as an immunogen.

9 Oct 2020

Synthetic group A streptogramin antibiotics that overcome Vat resistance (Nature)

Natural products serve as chemical blueprints for most antibiotics in clinical use. The evolutionary process by which these molecules arise is inherently accompanied by the co-evolution of resistance mechanisms that shorten the clinical lifetime of any given class of antibiotics. Virginiamycin acetyltransferase (Vat) enzymes are resistance proteins that provide protection against streptogramins, potent antibiotics against Gram-positive bacteria that inhibit the bacterial ribosome. Owing to the challenge of selectively modifying the chemically complex, 23-membered macrocyclic scaffold of group A streptogramins, analogues that overcome the resistance conferred by Vat enzymes have not been previously developed. Here we report the design, synthesis, and antibacterial evaluation of group A streptogramin antibiotics with extensive structural variability. Using cryo-electron microscopy and forcefield-based refinement, I.B.Seiple et al. characterize the binding of eight analogues to the bacterial ribosome at high resolution, revealing binding interactions that extend into the peptidyl tRNA-binding site and towards synergistic binders that occupy the nascent peptide exit tunnel. One of these analogues has excellent activity against several streptogramin-resistant strains of Staphylococcus aureus, exhibits decreased rates of acetylation in vitro, and is effective at lowering bacterial load in a mouse model of infection. Their results demonstrate that the combination of rational design and modular chemical synthesis can revitalize classes of antibiotics that are limited by naturally arising resistance mechanisms.

29 Sep 2020

Mechanism of ribosome shutdown by RsfS in Staphylococcus aureus revealed by integrative structural biology approach (Nat. Commun.)

For the sake of energy preservation, bacteria, upon transition to stationary phase, tone down their protein synthesis. This process is favored by the reversible binding of small stress- induced proteins to the ribosome to prevent unnecessary translation. One example is the conserved bacterial ribosome silencing factor (RsfS) that binds to uL14 protein onto the large ribosomal subunit and prevents its association with the small subunit. Here G. Yusupova, M, Yusupov et.al. describe the binding mode of Staphylococcus aureus RsfS to the large ribosomal subunit and present a 3.2 Å resolution cryo-EM reconstruction of the 50S-RsfS complex together with the crystal structure of uL14-RsfS complex solved at 2.3 Å resolution. The understanding of the detailed landscape of RsfS-uL14 interactions within the ribosome shed light on the mechanism of ribosome shutdown in the human pathogen S. aureus and might deliver a novel target for pharmacological drug development and treatment of bacterial infections.

29 Sep 2020

SYNERGISTIC ON AUXIN AND CYTOKININ 1 positively regulates growth and attenuates soil pathogen resistance (Nature)

Plants as non-mobile organisms constantly integrate varying environmental signals to flexibly adapt their growth and development. Local fluctuations in water and nutrient availability, sudden changes in temperature or other abiotic and biotic stresses can trigger changes in the growth of plant organs. Multiple mutually interconnected hormonal signaling cascades act as essential endogenous translators of these exogenous signals in the adaptive responses of plants. Although the molecular backbones of hormone transduction pathways have been identified, the mechanisms underlying their interactions are largely unknown. Here, using genome wide transcriptome profiling E. Benková et. al. identify an auxin and cytokinin cross-talk component; SYNERGISTIC ON AUXIN AND CYTOKININ 1 (SYAC1), whose expression in roots is strictly dependent on both of these hormonal pathways. They show that SYAC1 is a regulator of secretory pathway, whose enhanced activity interferes with deposition of cell wall components and can fine-tune organ growth and sensitivity to soil pathogens.

8 Sep 2020

Conformational Ensembles of an Intrinsically Disordered Protein Consistent with NMR, SAXS, and Single-Molecule FRET (J.Amer.Chem.Soc.)

Intrinsically disordered proteins (IDPs) have fluctuating heterogeneous conformations, which makes their structural characterization challenging. Although challenging, characterization of the conformational ensembles of IDPs is of great interest, since their conformational ensembles are the link between their sequences and functions. An accurate description of IDP conformational ensembles depends crucially on the amount and quality of the experimental data, how it is integrated, and if it supports a consistent structural picture. G-N.W. Gomes and C. Gradinaru et.al. used integrative modeling and validation to apply conformational restraints and assess agreement with the most common structural techniques for IDPs: Nuclear Magnetic Resonance (NMR) spectroscopy, Small-angle X-ray Scattering (SAXS), and single-molecule Förster Resonance Energy Transfer (smFRET). Agreement with such a diverse set of experimental data suggests that details of the generated ensembles can now be examined with a high degree of confidence. Using the disordered N-terminal region of the Sic1 protein as a test case, they examined relationships between average global polymeric descriptions and higher-moments of their distributions. To resolve apparent discrepancies between smFRET and SAXS inferences, they integrated SAXS data with NMR data and reserved the smFRET data for independent validation. Consistency with smFRET, which was not guaranteed a priori, indicates that, globally, the perturbative effects of NMR or smFRET labels on the Sic1 ensemble are minimal. Analysis of the ensembles revealed distinguishing features of Sic1, such as overall compactness and large end-to-end distance fluctuations, which are consistent with biophysical models of Sic1’s ultrasensitive binding to its partner Cdc4. Their results underscore the importance of integrative modeling and validation in generating and drawing conclusions from IDP conformational ensembles.

1 Sep 2020

In-cell architecture of an actively transcribing-translating expressome (Science)

Structural biology studies performed inside cells can capture molecular machines in action within their native context. In this work, we developed an integrative in-cell structural approach using the genome-reduced human pathogen Mycoplasma pneumoniae. J. Rappsilber et. al. combined whole-cell cross-linking mass spectrometry, cellular cryo–electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at subnanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. They pinpointed NusA at the interface between a NusG-bound elongating RNAP and the ribosome and propose that it can mediate transcription-translation coupling. Translation inhibition dissociated the expressome, whereas transcription inhibition stalled and rearranged it. Thus, the active expressome architecture requires both translation and transcription elongation within the cell.

27 Aug 2020

Half a century of amyloids: past, present and future (Chem. Rev.)

Amyloid diseases are global epidemics with profound health, social and economic implications and yet remain without a cure. This dire situation calls for research into the origin and pathological manifestations of amyloidosis to stimulate continued development of new therapeutics. In basic science and engineering, the cross-beta architecture has been a constant thread underlying the structural characteristics of pathological and functional amyloids, and realizing that amyloid structures can be both pathological and functional in nature has fueled innovations in artificial amyloids, whose use today ranges from water purification to 3D printing. At the conclusion of a half century since Eanes and Glenner's seminal study of amyloids in humans, this review commemorates the occasion by documenting the major milestones in amyloid research to date, from the perspectives of structural biology, biophysics, medicine, microbiology, engineering and nanotechnology. We also discuss new challenges and opportunities to drive this interdisciplinary field moving forward.

27 Aug 2020

NMR Methods for Structural Characterization of Protein-Protein Complexes (Front. Mol. Biosci.)

Protein-protein interactions and the complexes thus formed are critical elements in a wide variety of cellular events that require an atomic-level description to understand them in detail. Such complexes typically constitute challenging systems to characterize and drive the development of innovative biophysical methods. NMR spectroscopy techniques can be applied to extract atomic resolution information on the binding interfaces, intermolecular affinity, and binding-induced conformational changes in protein-protein complexes formed in solution, in the cell membrane, and in large macromolecular assemblies. Here Venditti V. et al. discuss experimental techniques for the characterization of protein-protein complexes in both solution NMR and solid-state NMR spectroscopy. The approaches include solvent paramagnetic relaxation enhancement and chemical shift perturbations (CSPs) for the identification of binding interfaces, and the application of intermolecular nuclear Overhauser effect spectroscopy and residual dipolar couplings to obtain structural constraints of protein-protein complexes in solution. Complementary methods in solid-state NMR are described, with emphasis on the versatility provided by heteronuclear dipolar recoupling to extract intermolecular constraints in differentially labeled protein complexes. The methods described are of particular relevance to the analysis of membrane proteins, such as those involved in signal transduction pathways, since they can potentially be characterized by both solution and solid-state NMR techniques, and thus outline key developments in this frontier of structural biology.

19 Aug 2020

Structure of human GABA(B) receptor in an inactive state (Nature)

The human GABA(B) receptor-a member of the class C family of G-protein-coupled receptors (GPCRs)-mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction. A unique GPCR that is known to require heterodimerization for function, the GABA(B) receptor has two subunits, GABA(B1) and GABA(B2), that are structurally homologous but perform distinct and complementary functions. GABA(B1) recognizes orthosteric ligands, while GABA(B2) couples with G proteins. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane domain and a cytoplasmic tail. Although the VFT heterodimer structure has been resolved, the structure of the full-length receptor and its transmembrane signalling mechanism remain unknown. Here O.B. Clarke, J. Frank, Q.R. Fan et al. present a near full-length structure of the GABA(B) receptor at atomic resolution, captured in an inactive state by cryo-electron microscopy. Their structure reveals several ligands that preassociate with the receptor, including two large endogenous phospholipids that are embedded within the transmembrane domains to maintain receptor integrity and modulate receptor function. They also identify a previously unknown heterodimer interface between transmembrane helices 3 and 5 of both subunits, which serves as a signature of the inactive conformation. A unique 'intersubunit latch' within this transmembrane interface maintains the inactive state, and its disruption leads to constitutive receptor activity. The structure of the GABA(B) receptor in an inactive state reveals, amongst other features, a latch between the two subunits that locks the transmembrane domain interface, and the presence of large phospholipids that may modulate receptor function.