11 Jul 2022

Atomic-Resolution Structure of SARS-CoV-2 Nucleocapsid Protein N-Terminal Domain (J. Amer.Chem. Soc.)

The nucleocapsid (N) protein is one of the four structural proteins of the SARS-CoV-2 virus and plays a crucial role in viral genome organization and, hence, replication and pathogenicity. The N-terminal domain (N^NTD) binds to the genomic RNA and thus comprises a potential target for inhibitor and vaccine development. We determined the atomic-resolution structure of crystalline N^NTD by integrating solid-state magic angle spinning (MAS) NMR and X-ray diffraction. Our combined approach provides atomic details of protein packing interfaces as well as information about flexible regions as the N- and C-termini and the functionally important RNA binding, β-hairpin loop. In addition, ultrafast (100 kHz) MAS ¹H-detected experiments permitted the assignment of side-chain proton chemical shifts not available by other means. The present structure offers guidance for designing therapeutic interventions against the SARS-CoV-2 infection.

11 Jul 2022

Defining the substrate envelope of SARS-CoV-2 main protease to predict and avoid drug resistance (Nature Communications)

Coronaviruses can evolve and spread rapidly to cause severe disease morbidity and mortality, as exemplified by SARS-CoV-2 variants of the COVID-19 pandemic. Although currently available vaccines remain mostly effective against SARS-CoV-2 variants, additional treatment strategies are needed. Inhibitors that target essential viral enzymes, such as proteases and polymerases, represent key classes of antivirals. However, clinical use of antiviral therapies inevitably leads to emergence of drug resistance. In this study we implemented a strategy to pre-emptively address drug resistance to protease inhibitors targeting the main protease (M^pro) of SARS-CoV-2, an essential enzyme that promotes viral maturation. We solved nine high-resolution cocrystal structures of SARS-CoV-2 M^pro bound to substrate peptides and six structures with cleavage products. These structures enabled us to define the substrate envelope of M^pro, map the critical recognition elements, and identify evolutionarily vulnerable sites that may be susceptible to resistance mutations that would compromise binding of the newly developed M^pro inhibitors. Our results suggest strategies for developing robust inhibitors against SARS-CoV-2 that will retain longer-lasting efficacy against this evolving viral pathogen.

11 Jul 2022

The mechanisms of catalysis and ligand binding for the SARS-CoV-2 NSP3 macrodomain from neutron and x-ray diffraction at room temperature (Science Advances)

The nonstructural protein 3 (NSP3) macrodomain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Mac1) removes adenosine diphosphate (ADP) ribosylation posttranslational modifications, playing a key role in the immune evasion capabilities of the virus responsible for the coronavirus disease 2019 pandemic. Here, we determined neutron and x-ray crystal structures of the SARS-CoV-2 NSP3 macrodomain using multiple crystal forms, temperatures, and pHs, across the apo and ADP-ribose–bound states. We characterize extensive solvation in the Mac1 active site and visualize how water networks reorganize upon binding of ADP-ribose and non-native ligands, inspiring strategies for displacing waters to increase the potency of Mac1 inhibitors. Determining the precise orientations of active site water molecules and the protonation states of key catalytic site residues by neutron crystallography suggests a catalytic mechanism for coronavirus macrodomains distinct from the substrate-assisted mechanism proposed for human MacroD2. These data provoke a reevaluation of macrodomain catalytic mechanisms and will guide the optimization of Mac1 inhibitors.

8 Aug 2023

Integrated NMR/Molecular Dynamics Determination of the Ensemble Conformation of a Thermodynamically Stable CUUG RNA Tetraloop (Journal of the American Chemical Society)

Both experimental and theoretical structure determinations of RNAs have remained challenging due to the intrinsic dynamics of RNAs. We report here an integrated nuclear magnetic resonance/molecular dynamics (NMR/MD) structure determination approach to describe the dynamic structure of the CUUG tetraloop. We show that the tetraloop undergoes substantial dynamics, leading to averaging of the experimental data. These dynamics are particularly linked to the temperature-dependent presence of a hydrogen bond within the tetraloop. Interpreting the NMR data by a single structure represents the low-temperature structure well but fails to capture all conformational states occurring at a higher temperature. We integrate MD simulations, starting from structures of CUUG tetraloops within the Protein Data Bank, with an extensive set of NMR data, and provide a structural ensemble that describes the dynamic nature of the tetraloop and the experimental NMR data well. We thus show that one of the most stable and frequently found RNA tetraloops displays substantial dynamics, warranting such an integrated structural approach.

24 Jul 2023

Cannabidiol inhibits Nav channels through two distinct binding sites (Nature Communications)

Cannabidiol (CBD), a major non-psychoactive phytocannabinoid in cannabis, is an effective treatment for some forms of epilepsy and pain. At high concentrations, CBD interacts with a huge variety of proteins, but which targets are most relevant for clinical actions is still unclear. Here we show that CBD interacts with Na_v1.7 channels at sub-micromolar concentrations in a state-dependent manner. Electrophysiological experiments show that CBD binds to the inactivated state of Na_v1.7 channels with a dissociation constant of about 50 nM. The cryo-EM structure of CBD bound to Na_v1.7 channels reveals two distinct binding sites. One is in the IV-I fenestration near the upper pore. The other binding site is directly next to the inactivated “wedged” position of the Ile/Phe/Met (IFM) motif on the short linker between repeats III and IV, which mediates fast inactivation. Consistent with producing a direct stabilization of the inactivated state, mutating residues in this binding site greatly reduced state-dependent binding of CBD. The identification of this binding site may enable design of compounds with improved properties compared to CBD itself.

24 Jul 2023

Combining Solid-State NMR with Structural and Biophysical Techniques to Design Challenging Protein-Drug Conjugates (Angewandte Chemie Int. Ed.)

Several protein-drug conjugates are currently being used in cancer therapy. These conjugates rely on cytotoxic organic compounds that are covalently attached to the carrier proteins or that interact with them via non-covalent interactions. Human transthyretin (TTR), a physiological protein, has already been identified as a possible carrier protein for the delivery of cytotoxic drugs. Here we show the structure-guided development of a new stable cytotoxic molecule based on a known strong binder of TTR and a well-established anticancer drug. This example is used to demonstrate the importance of the integration of multiple biophysical and structural techniques, encompassing microscale thermophoresis, X-ray crystallography and NMR. In particular, we show that solid-state NMR has the ability to reveal effects caused by ligand binding which are more easily relatable to structural and dynamical alterations that impact the stability of macromolecular complexes.

24 Jul 2023

Dynamics of Membranous Cholesterol are Coupled (Journal of the American Chemical Society)

Cholesterol promotes the structural integrity of the fluid cell membrane and interacts dynamically with many membrane proteins to regulate function. Understanding site-resolved cholesterol structural dynamics is thus important. This long-standing challenge has thus far been addressed, in part, by selective isotopic labeling approaches. Here we present a new 3D solid-state NMR (SSNMR) experiment utilizing scalar ¹³C–¹³C polarization transfer and recoupling of the ¹H–¹³C interactions in order to determine average dipolar couplings for all ¹H–¹³C vectors in uniformly ¹³C-enriched cholesterol. The experimentally determined order parameters (OP) agree exceptionally well with molecular dynamics (MD) trajectories and reveal coupling among several conformational degrees of freedom in cholesterol molecules. Quantum chemistry shielding calculations further support this conclusion and specifically demonstrate that ring tilt and rotation are coupled to changes in tail conformation and that these coupled segmental dynamics dictate the orientation of cholesterol. These findings advance our understanding of physiologically relevant dynamics of cholesterol, and the methods that revealed them have broader potential to characterize how structural dynamics of other small molecules impact their biological functions.

17 Jul 2023

RNA polymerase drives ribonucleotide excision DNA repair in E. coli (Cell)

Ribonuclease HII (RNaseHII) is the principal enzyme that removes misincorporated ribonucleoside monophosphates (rNMPs) from genomic DNA. Here, we present structural, biochemical, and genetic evidence demonstrating that ribonucleotide excision repair (RER) is directly coupled to transcription. Affinity pull-downs and mass-spectrometry-assisted mapping of in cellulo inter-protein cross-linking reveal the majority of RNaseHII molecules interacting with RNA polymerase (RNAP) in E. coli. Cryoelectron microscopy structures of RNaseHII bound to RNAP during elongation, with and without the target rNMP substrate, show specific protein-protein interactions that define the transcription-coupled RER (TC-RER) complex in engaged and unengaged states. The weakening of RNAP-RNaseHII interactions compromises RER in vivo. The structure-functional data support a model where RNaseHII scans DNA in one dimension in search for rNMPs while “riding” the RNAP. We further demonstrate that TC-RER accounts for a significant fraction of repair events, thereby establishing RNAP as a surveillance “vehicle” for detecting the most frequently occurring replication errors.

17 Jul 2023

MicroED in drug discovery (Curr. Opin. Struct. Biol.)

The cryo-electron microscopy (cryo-EM) method microcrystal electron diffraction (MicroED) was initially described in 2013 and has recently gained attention as an emerging technique for research in drug discovery. As compared to other methods in structural biology, MicroED provides many advantages deriving from the use of nanocrystalline material for the investigations. Here, we review the recent advancements in the field of MicroED and show important examples of small molecule, peptide and protein structures that has contributed to the current development of this method as an important tool for drug discovery.

17 Jul 2023

Visualizing everything, everywhere, all at once: Cryo-EM and the new field of structureomics (Curr. Opin. Struct. Biol.)

Twenty years ago, the release of the first draft of the human genome sequence instigated a paradigm shift in genomics and molecular biology. Arguably, structural biology is entering an analogous era, with availability of an experimentally determined or predicted molecular model for almost every protein-coding gene from many genomes—producing a reference “structureome”. Structural predictions require experimental validation and not all proteins conform to a single structure, making any reference structureome necessarily incomplete. Despite these limitations, a reference structureome can be used to characterize cell state in more detail than by quantifying sequence or expression levels alone. Cryogenic electron microscopy (cryo-EM) is a method that can generate atomic resolution views of molecules and cells frozen in place. In this perspective I consider how emerging cryo-EM methods are contributing to the new field of structureomics.

10 Jul 2023

Adenosine A(2A) Receptor (A(2A)AR) Ligand Screening Using the F-19-NMR Probe FPPA

The binding affinity of G protein-coupledreceptor (GPCR)ligandsis customarily measured by radio-ligand competition experiments. As an alternative approach, F-19 nuclear magnetic resonancespectroscopy (F-19-NMR) is used for the screening of small-moleculelead compounds in drug discovery; the two methods are complementary in that the measurements are performed with widely different experimental conditions. Here, we used the structure of the A(2A) adenosinereceptor (A(2A)AR) complex with V-2006 (3-(4-amino-3-methylbenzyl)-7-(furan-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine)as the basis for the design of a fluorine-containing probe molecule, FPPA (4-(furan-2-yl)-7-(4-(trifluoromethyl)benzyl)-7H-pyrrolo[2,3-d]pyramidin-2-amine), forbinding studies with A(2A)AR. A protocol of experimental conditions for drug screening and measurements of drug binding affinities using 1D F-19-NMR observation of FPPA is validated with studies of known A(2A)AR ligands. F-19-NMR with FPPA is thus found to be a robust approach for the discovery of ligands with new core structures, which will expand the libraries of A(2A)AR-targeting drug candidates.