Bacterial J-Domains with C-Terminal Tags Contact the Substrate Binding Domain of DnaK and Sequester Chaperone Activity.

Functional interactions between the molecular chaperone DnaK and cofactor J-proteins (DnaJs), as well as their homologs, are crucial to the maintenance of proteostasis across cell types. In the bacterial pathogen Mycobacterium tuberculosis, DnaK-DnaJ interactions are essential for cell growth and represent potential targets for antibiotic or adjuvant development. While the N-terminal J-domains of J-proteins are known to form important contacts with DnaK, C-terminal domains have varied roles. Here, we have studied the effect of adding C-terminal tags to N-terminal J-domain truncations of mycobacterial DnaJ1 and DnaJ2 to promote additional interactions with DnaK. We found that His6 tags uniquely promote binding to additional sites in the substrate binding domain at the C-terminus of DnaK. Other C-terminal tags attached to J-domains, even peptides known to interact with DnaK, do not produce the same effects. Expression of C-terminally modified DnaJ1 or DnaJ2 J-domains in mycobacterial cells suppresses chaperone activity following proteotoxic stress, which is exaggerated in the presence of a small-molecule DnaK inhibitor. Hence, this work uncovers genetically encodable J-protein variants that may be used to study chaperone-cofactor interactions in other organisms.

A Repurposed Drug Interferes with Nucleic Acid to Inhibit the Dual Activities of Coronavirus Nsp13.

The recent pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlighted a critical need to discover more effective antivirals. While therapeutics for SARS-CoV-2 exist, its nonstructural protein 13 (Nsp13) remains a clinically untapped target. Nsp13 is a helicase responsible for unwinding double-stranded RNA during viral replication and is essential for propagation. Like other helicases, Nsp13 has two active sites: a nucleotide binding site that hydrolyzes nucleoside triphosphates (NTPs) and a nucleic acid binding channel that unwinds double-stranded RNA or DNA. Targeting viral helicases with small molecules, as well as the identification of ligand binding pockets, have been ongoing challenges, partly due to the flexible nature of these proteins. Here, we use a virtual screen to identify ligands of Nsp13 from a collection of clinically used drugs. We find that a known ion channel inhibitor, IOWH-032, inhibits the dual ATPase and helicase activities of SARS-CoV-2 Nsp13 at low micromolar concentrations. Kinetic and binding assays, along with computational and mutational analyses, indicate that IOWH-032 interacts with the RNA binding interface, leading to displacement of nucleic acid substrate, but not bound ATP. Evaluation of IOWH-032 with microbial helicases from other superfamilies reveals that it is selective for coronavirus Nsp13. Furthermore, it remains active against mutants representative of observed SARS-CoV-2 variants. Overall, this work provides a new inhibitor for Nsp13 and provides a rationale for a recent observation that IOWH-032 lowers SARS-CoV-2 viral loads in human cells, setting the stage for the discovery of other potent viral helicase modulators.

Four millimeter spherical rotors spinning at 28 kHz with double-saddle coils for cross polarization NMR.

Spherical rotors in magic angle spinning (MAS) experiments have significant advantages over traditional cylindrical rotors including simplified spinning implementation, easy sample exchange, more efficient microwave coupling for dynamic nuclear polarization (DNP), and feasibility of downscaling to access higher spinning frequencies. Here, we implement spherical rotors with 4 mm outside diameter (o.d.) and demonstrate spinning >28 kHz using a single aperture for spinning gas. We show a modified stator geometry to improve fiber optic detection, increase NMR filling factor, and improve alignment for sample exchange and microwave irradiation. Higher NMR Rabi frequencies were obtained using smaller radiofrequency (RF) coils on small-diameter spherical rotors, compared to our previous implementation of MAS spheres with an o.d. of 9.5 mm. We report nutation fields of 110 kHz on 13C with 820 W of input power and 100 kHz on 1H with 800 W of input power. Proton decoupling fields of 78 kHz were applied over 20 ms of signal acquisition without any sign of arcing. Compared to our initial demonstration of a split coil for 9.5 mm spheres, this current implementation of a double-saddle coil inductor for 4 mm spheres not only intensifies the RF fields, but also improves RF homogeneity. We achieve an 810°/90° nutation intensity ratio of 0.84 at 300.197 MHz (1H). We also show electromagnetic simulations predicting a nearly 3-fold improvement in electron Rabi frequency of 0.99 MHz (with 4 mm spheres) compared to 0.38 MHz (with 3.2 mm cylinders), with 5 W of incident microwave power. Further improvements in magnetic resonance spin control are expected as RF inductors and microwave coupling are optimized for spherical rotors and scaled down to the micron scale.