NMR Spectroscopy of the Main Protease of SARS-CoV-2 and Fragment-Based Screening Identify Three Protein Hotspots and an Antiviral Fragment

 

NMR Spectroscopy of the Main Protease of SARS-CoV-2 and Fragment-Based Screening Identify Three Protein Hotspots and an Antiviral Fragment

The main protease (3CLp) of the SARS-CoV-2, the causative agent for the COVID-19 pandemic, is one of the main targets for drug development. To be active, 3CLp relies on a complex interplay between dimerization, active site flexibility, and allosteric regulation. The deciphering of these mechanisms is a crucial step to enable the search for inhibitors. In this context, using NMR spectroscopy, we studied the conformation of dimeric 3CLp from the SARS-CoV-2 and monitored ligand binding, based on NMR signal assignments. We performed a fragment-based screening that led to the identification of 38 fragment hits. Their binding sites showed three hotspots on 3CLp, two in the substrate binding pocket and one at the dimer interface. F01 is a non-covalent inhibitor of the 3CLp and has antiviral activity in SARS-CoV-2 infected cells. This study sheds light on the complex structure-function relationships of 3CLp and constitutes a strong basis to assist in developing potent 3CLp inhibitors.

Published: September 27, 2021 DOI: https://doi.org/10.1002/anie.202109965

 

Phosphorylation and O-GlcNAcylation of the PHF-1 Epitope of Tau Protein Induce Local Conformational Changes of the C-Terminus and Modulate Tau Self-Assembly Into Fibrillar Aggregates

 

Phosphorylation and O-GlcNAcylation of the PHF-1 Epitope of Tau Protein Induce Local Conformational Changes of the C-Terminus and Modulate Tau Self-Assembly Into Fibrillar Aggregates

Phosphorylation of the neuronal microtubule-associated Tau protein plays a critical role in the aggregation process leading to the formation of insoluble intraneuronal fibrils within Alzheimer’s disease (AD) brains. In recent years, other posttranslational modifications (PTMs) have been highlighted in the regulation of Tau (dys)functions. Among these PTMs, the Oβ-linked N-acetylglucosaminylation (O-GlcNAcylation) modulates Tau phosphorylation and aggregation. We here focus on the role of the PHF-1 phospho-epitope of Tau C-terminal domain that is hyperphosphorylated in AD (at pS396/pS404) and encompasses S400 as the major O-GlcNAc site of Tau while two additional O-GlcNAc sites were found in the extreme C-terminus at S412 and S413. Using high resolution NMR spectroscopy, we showed that the O-GlcNAc glycosylation reduces phosphorylation of PHF-1 epitope by GSK3β alone or after priming by CDK2/cyclin A. Furthermore, investigations of the impact of PTMs on local conformation performed in small peptides highlight the role of S404 phosphorylation in inducing helical propensity in the region downstream pS404 that is exacerbated by other phosphorylations of PHF-1 epitope at S396 and S400, or O-GlcNAcylation of S400. Finally, the role of phosphorylation and O-GlcNAcylation of PHF-1 epitope was probed in in-vitro fibrillization assays in which O-GlcNAcylation slows down the rate of fibrillar assembly while GSK3β phosphorylation stimulates aggregation counteracting the effect of glycosylation.

Open Access ⏐ Published: June 21, 2021 DOI: 10.3389/fnmol.2021.661368

Take your PIC

 

Take your PIC

Cryo-electron microscopy has enabled unprecedented progress in the quest to reveal the structure of the whole transcription preinitiation complex. Four recent studies pave the way for a complete description of how transcription is initiated at near-atomic level.

Published: June 5, 2021 DOI: https://doi.org/10.1016/j.tibs.2021.05.008

Probing Long-Range Anisotropic Interactions: a General and Sign-Sensitive Strategy to Measure 1H- 1H Residual Dipolar Couplings as a Key Advance for Organic Structure Determination

 

Probing Long-Range Anisotropic Interactions: a General and Sign-Sensitive Strategy to Measure 1H- 1H Residual Dipolar Couplings as a Key Advance for Organic Structure Determination

Residual dipolar couplings (RDCs) are amongst the most powerful NMR parameters for organic structure elucidation. In order to maximize their effectiveness in increasingly complex cases such as flexible compounds, a maximum of RDCs between nuclei sampling a large distribution of orientations is needed, including sign information. For this, the easily accessible one-bond 1H–13C RDCs alone often fall short. Long-range 1H–1H RDCs are both abundant and typically sample highly complementary orientations, but accessing them in a sign-sensitive way has been severely obstructed due to the overflow of 1H–1H couplings. Here, we present a generally applicable strategy that allows the measurement of a large number of 1H–1H RDCs, including their signs, which is based on a combination of an improved PSYCHEDELIC method and a new selective constant-time β-COSY experiment. The potential of 1H–1H RDCs to better determine molecular alignment and to discriminate between enantiomers and diastereomers is demonstrated.

Published: February 11, 2020 DOI:  https://doi.org/10.1002/anie.201915278