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Highlights of Coronavirus Structural Studies

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Reader's Corner Archive

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.

27 Oct 2020

Retrieving functional pathways of biomolecules from single-particle snapshots (Nat. Commun.)

A primary reason for the intense interest in structural biology is the fact that knowledge of structure can elucidate macromolecular functions in living organisms. Sustained effort has resulted in an impressive arsenal of tools for determining the static structures. But under physiological conditions, macromolecules undergo continuous conformational changes, a subset of which are functionally important. Techniques for capturing the continuous conformational changes underlying function are essential for further progress. Here, des Georges, Singharoy, Frank, and Ourmazd et. al. present chemically-detailed conformational movies of biological function, extracted data-analytically from experimental single-particle cryo-electron microscopy (cryo-EM) snapshots of ryanodine receptor type 1 (RyR1), a calcium-activated calcium channel engaged in the binding of ligands. The functional motions differ substantially from those inferred from static structures in the nature of conformationally active structural domains, the sequence and extent of conformational motions, and the way allosteric signals are transduced within and between domains. Their approach highlights the importance of combining experiment, advanced data analysis, and molecular simulations. There is a great interest in retrieving functional pathways from cryo-EM single-particle data. Here, the authors present an approach that combines cryo-EM with advanced data-analytical methods and molecular dynamics simulations to reveal the functional pathways traversed on experimentally derived energy landscapes using the ryanodine receptor type 1 as an example.

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.

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