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

Cryo-EM analysis of a membrane protein embedded in the liposome (PNAS)

Membrane proteins (MPs) used to be the most difficult targets for structural biology when X-ray crystallography was the mainstream approach. With the resolution revolution of single-particle electron cryo-microscopy (cryo-EM), rapid progress has been made for structural elucidation of isolated MPs. The next challenge is to preserve the electrochemical gradients and membrane curvature for a comprehensive structural elucidation of MPs that rely on these chemical and physical properties for their biological functions. Toward this goal, here Fan, X., Yan, N. et.al. present a convenient workflow for cryo-EM structural analysis of MPs embedded in liposomes, using the well-characterized AcrB as a prototype. Combining optimized proteoliposome isolation, cryo-sample preparation on graphene grids, and an efficient particle selection strategy, the three-dimensional (3D) reconstruction of AcrB embedded in liposomes was obtained at 3.9 angstrom resolution. The conformation of the homotrimeric AcrB remains the same when the surrounding membranes display different curvatures. Their approach, which can be widely applied to cryo-EM analysis of MPs with distinctive soluble domains, lays out the foundation for cryo-EM analysis of integral or peripheral MPs whose functions are affected by transmembrane electrochemical gradients or/and membrane curvatures.

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

Evidence that the TRPV1 S1-S4 membrane domain contributes to thermosensing (Nat. Commun.)

Sensing and responding to temperature is crucial in biology. The TRPV1 ion channel is a well- studied heat-sensing receptor that is also activated by vanilloid compounds, including cap- saicin. Despite significant interest, the molecular underpinnings of thermosensing have remained elusive. The TRPV1 S1-S4 membrane domain couples chemical ligand binding to the pore domain during channel gating. Here W.D.Van Horn e.al. show that the S1-S4 domain also significantly contributes to thermosensing and couples to heat-activated gating. Evaluation of the isolated human TRPV1 S1-S4 domain by solution NMR, far-UV CD, and intrinsic fluorescence shows that this domain undergoes a non-denaturing temperature-dependent transition with a high thermosensitivity. Further NMR characterization of the temperature-dependent conformational changes suggests the contribution of the S1-S4 domain to thermosensing shares features with known coupling mechanisms between this domain with ligand and pH activation. Taken together, this study shows that the TRPV1 S1-S4 domain contributes to TRPV1 temperature-dependent activation.

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.