Structures of SAS-6 coiled coil hold implications for the polarity of the centriolar cartwheel

Centrioles are eukaryotic organelles that template the formation of cilia and flagella, as well as organize the microtubule network and the mitotic spindle in animal cells. Centrioles have proximal-distal polarity and a 9-fold radial symmetry imparted by a likewise symmetrical central scaffold, the cartwheel. The spindle assembly abnormal protein 6 (SAS-6) self-assembles into 9-fold radially symmetric ring-shaped oligomers that stack via an unknown mechanism to form the cartwheel. Here, we uncover a homo-oligomerization interaction mediated by the coiled-coil domain of SAS-6. Crystallographic structures of Chlamydomonas reinhardtii SAS-6 coiled-coil complexes suggest this interaction is asymmetric, thereby imparting polarity to the cartwheel. Using a cryoelectron microscopy (cryo-EM) reconstitution assay, we demonstrate that amino acid substitutions disrupting this asymmetric association also impair SAS-6 ring stacking. Our work raises the possibility that the asymmetric interaction inherent to SAS-6 coiled-coil provides a polar element for cartwheel assembly, which may assist the establishment of the centriolar proximal-distal axis

A COVID moonshot: assessment of ligand binding to the SARS-CoV-2 main protease by saturation transfer difference NMR spectroscopy

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological cause of the coronavirus disease 2019, for which no efective antiviral therapeutics are available. The SARS-CoV-2 main protease (Mpro) is essential for viral replication and constitutes a promising therapeutic target. Many eforts aimed at deriving efective Mpro inhibitors are currently underway, including an international open-science discovery project, codenamed COVID Moonshot. As part of COVID Moonshot, we used saturation transfer diference nuclear magnetic resonance (STD-NMR) spectroscopy to assess the binding of putative Mpro ligands to the viral protease, including molecules identifed by crystallographic fragment screening and novel compounds designed as Mpro inhibitors. In this manner, we aimed to complement enzymatic activity assays of Mpro performed by other groups with information on ligand afnity. We have made the Mpro STD-NMR data publicly available. Here, we provide detailed information on the NMR protocols used and challenges faced, thereby placing these data into context. Our goal is to assist the interpretation of Mpro STD-NMR data, thereby accelerating ongoing drug design eforts

Allosteric inhibition of the SARS‐CoV‐2 main protease – insights from mass spectrometry‐based assays

Following translation of the SARS‐CoV‐2 RNA genome into two viral polypeptides, the main protease M pro cleaves at eleven sites to release non‐structural proteins required for viral replication. M Pro is an attractive target for antiviral therapies to combat the coronavirus‐2019 disease (COVID‐19). Here, we have used native mass spectrometry (MS) to characterize the functional unit of M pro . Analysis of the monomer‐dimer equilibria reveals a dissociation constant of K d = 0.14 ± 0.03 µM, revealing M Pro has a strong preference to dimerize in solution. Developing an MS‐based kinetic assay we then characterized substrate turnover rates by following temporal changes in the enzyme‐substrate complexes, which are effectively “flash‐frozen” as they transition from solution to the gas phase. We screened small molecules, that bind distant from the active site, for their ability to modulate activity. These compounds, including one proposed to disrupt the catalytically active dimer, slow the rate of substrate processing by ~35%. This information was readily obtained and, together with analysis of the x‐ray crystal structures of these enzyme‐small molecule complexes, provides a starting point for the development of more potent molecules that allosterically regulate M Pro activity