13 Apr 2021

NMR spectroscopy captures the essential role of dynamics in regulating biomolecular function (Cell)

Biomolecules are in constant motion. To understand how they function, and why malfunctions can cause disease, it is necessary to describe their three-dimensional structures in terms of dynamic conformational ensembles. Here, L. Kay and T.R. Alderson demonstrate how nuclearmagnetic resonance (NMR) spectroscopy provides an essential, dynamic view of structural biology that captures biomolecular motions at atomic resolution. They focus on examples that emphasize the diversity of biomolecules and biochemical applications that are amenable to NMR, such as elucidating functional dynamics in large molecular machines, characterizing transient conformations implicated in the onset of disease, and obtaining atomic-level descriptions of intrinsically disordered regions that make weak interactions involved in liquid-liquid phase separation. Finally, theydiscuss the pivotal role that NMR has played in driving forward our understanding of the biomolecular dynamics-function paradigm.

29 Mar 2021

A 'Build and Retrieve' methodology to simultaneously solve cryo-EM structures of membrane proteins (Nat. Methods)

Single-particle cryo-electron microscopy (cryo-EM) has become a powerful technique in the field of structural biology. However, the inability to reliably produce pure, homogeneous membrane protein samples hampers the progress of their structural determination. Here, E. W. Yu et. al. develop a bottom-up iterative method, Build and Retrieve (BaR), that enables the identification and determination of cryo-EM structures of a variety of inner and outer membrane proteins, including membrane protein complexes of different sizes and dimensions, from a heterogeneous, impure protein sample. They also use the BaR methodology to elucidate structural information from Escherichia coli K12 crude membrane and raw lysate. The findings demonstrate that it is possible to solve high-resolution structures of a number of relatively small (<100 kDa) and less abundant (<10%) unidentified membrane proteins within a single, heterogeneous sample. Importantly, these results highlight the potential of cryo-EM for systems structural proteomics.

29 Mar 2021

Unravelling the structural complexity of glycolipids with cryogenic infrared spectroscopy (Nat. Commun.)

Glycolipids are complex glycoconjugates composed of a glycan headgroup and a lipid moiety. Their modular biosynthesis creates a vast amount of diverse and often isomeric structures, which fulfill highly specific biological functions. To date, no gold-standard analytical technique can provide a comprehensive structural elucidation of complex glycolipids, and insufficient tools for isomer distinction can lead to wrong assignments. Herein K. Pagel et. al. use cryogenic gas-phase infrared spectroscopy to systematically investigate different kinds of isomerism in immunologically relevant glycolipids. They show that all structural features, including isomeric glycan headgroups, anomeric configurations and different lipid moieties, can be unambiguously resolved by diagnostic spectroscopic fingerprints in a narrow spectral range. The results allow for the characterization of isomeric glycolipid mixtures and biological applications.

29 Mar 2021

Current limitations to high-resolution structure determination by single-particle cryoEM (Q. Rev. Biophys.)

CryoEM has become the method of choice for determining the structure of large macromolecular complexes in multiple conformations, at resolutions where unambiguous atomic models can be built. Two effects that have limited progress in single-particle cryoEM are (i) beam- induced movement during image acquisition and (ii) protein adsorption and denaturation at the air-water interface during specimen preparation. While beam-induced movement now appears to have been resolved by all-gold specimen support grids with very small holes, surface effects at the air-water interface are a persistent problem. Strategies to overcome these effects include the use of alternative support films and new techniques for specimen deposition. E. D’Imprima and W. Kühlbrandt examine the future potential of recording perfect images of biological samples for routine structure determination at atomic resolution.

18 Mar 2021

Base-pair resolution analysis of the effect of supercoiling on DNA flexibility and major groove recognition by triplex-forming oligonucleotides (Nature Communications)

In the cell, DNA is arranged into highly organised and topologically constrained (supercoiled) structures. It remains unclear how this supercoiling affects the detailed double-helical structure of DNA, largely because of limitations in spatial resolution of the available biophysical tools. Here, A.L.B. Pyne, A. Noy, S. A. Harris et. al. overcome these limitations, by a combination of atomic force microscopy (AFM) and atomistic molecular dynamics (MD) simulations, to resolve structures of negatively supercoiled DNA minicircles at base-pair resolution. They observe that negative superhelical stress induces local variation in the canonical B-form DNA structure by introducing kinks and defects that affect global minicircle structure and flexibility. They probe how these local and global conformational changes affect DNA interactions through the binding of triplex-forming oligonucleotides to DNA minicircles. They show that the energetics of triplex formation is governed by a delicate balance between electrostatics and bonding interactions. Their results provide mechanistic insight into how DNA supercoiling can affect molecular recognition, that may have broader implications for DNA interactions with other molecular species.

12 Mar 2021

Recognition of non-CpG repeats in Alu and ribosomal RNAs by the Z-RNA binding domain of ADAR1 induces A-Z junctions (Nature Communications)

Adenosine-to-inosine (A-to-I) editing of eukaryotic cellular RNAs is essential for protection against auto-immune disorders. Editing is carried out by ADAR1, whose innate immune response-specific cytoplasmic isoform possesses a Z-DNA binding domain (Zα) of unknown function. Zα also binds to CpG repeats in RNA, which are a hallmark of Z-RNA formation. Unexpectedly, Zα has been predicted — and in some cases even shown — to bind to specific regions within mRNA and rRNA devoid of such repeats. Here, Q. Vicens, B. Vögeli et. al. use NMR, circular dichroism, and other biophysical approaches to demonstrate and characterize the binding of Zα to mRNA and rRNA fragments. Their results reveal a broad range of RNA sequences that bind to Zα and adopt Z-RNA conformations. Binding is accompanied by destabilization of neighbouring A-form regions which is similar in character to what has been observed for B-Z- DNA junctions. The binding of Zα to non-CpG sequences is specific, cooperative and occurs with an affinity in the low micromolar range. This work allows them to propose a model for how Zα could influence the RNA binding specificity of ADAR1.