Cryo-EM structures of the tubulin cofactors reveal the molecular basis for the biogenesis of alpha/beta-tubulin.

Microtubule polarity and dynamic polymerization originate from the self-association properties of the a-tubulin heterodimer. For decades, it has remained poorly understood how the tubulin cofactors, TBCD, TBCE, TBCC, and the Arl2 GTPase mediate a-tubulin biogenesis from α- and ß-tubulins. Here, we use cryogenic electron microscopy to determine structures of tubulin cofactors bound to αß-tubulin. These structures show that TBCD, TBCE, and Arl2 form a heterotrimeric cage-like TBC-DEG assembly around the a-tubulin heterodimer. TBCD wraps around Arl2 and almost entirely encircles -tubulin, while TBCE forms a lever arm that anchors along the other end of TBCD and rotates α-tubulin. Structures of the TBC-DEG-αß-tubulin assemblies bound to TBCC reveal the clockwise rotation of the TBCE lever that twists a-tubulin by pulling its C-terminal tail while TBCD holds -tubulin in place. Altogether, these structures uncover transition states in αß-tubulin biogenesis, suggesting a vise-like mechanism for the GTP-hydrolysis dependent a-tubulin biogenesis mediated by TBC-DEG and TBCC. These structures provide the first evidence of the critical functions of the tubulin cofactors as enzymes that regulate the invariant organization of αß-tubulin, by catalyzing α- and ß-tubulin assembly, disassembly, and subunit exchange which are crucial for regulating the polymerization capacities of αß-tubulins into microtubules.

Atomic structure of the regulatory TGS domain of Rel protein from Mycobacterium tuberculosis and its interaction with deacylated tRNA.

The stringent response is critical for the survival of Mycobacterium tuberculosis (Mtb) under nutrient starvation. The mechanism is mediated by a GTP pyrophosphokinase known as Rel, containing N-terminal synthetase and hydrolase domains and C-terminal regulatory domains, which include the TGS domain (ThrRS, GTPase, and SpoT proteins) that has been proposed to activate the synthetase domain via interaction with deacylated tRNA. Here, we present the NMR solution structure of the Mtb Rel TGS domain (MtRel TGS), consisting of five antiparallel ß-strands and one helix-loop-helix motif. The interaction of MtRel TGS with deacylated tRNA is shown, indicating the critical amino acids of MtRel TGS in tRNA binding, and presenting the first structural evidence of MtRel TGS binding to deacylated tRNA in solution in the absence of the translational machinery.

Emerging Therapeutic Approaches to COVID-19.

Coronavirus disease (COVID-19) is caused by a novel severe acute respiratory syndrome coronavirus (SARS-CoV-2), which is a positive single-stranded RNA virus having a large genome ~30 kb. SARSCoV- 2 is zoonotic and highly contagious, causing severe pneumonia-like symptoms. The efficacy of the different potential drug and drug candidates against COVID-19 has been investigated, which are under various stages of clinical trials. The drugs effective against SARS, and Middle east respiratory syndrome (MERS), have been proposed to have a high potential for the treatment of COVID-19. Here, we selected plant-based materials implicated in the prevention and therapy of COVID-19. The plant produces secondary metabolites in response to viral infection. Different classes of secondary metabolites have different mechanisms to counter virus attacks. Many nanomaterials produced by carbohydrates and lipids have been exploited for their in-vitro and in-vivo delivery of antiviral therapeutics. The vaccine has shown impressive results in producing antibodies against SARS-CoV2 and has been evaluated for safety, tolerance, and preliminary immunogenicity. Similarly, DNA/RNA-based therapy has shown high clinical significance. Various forms of vitamins, minerals, herbs, and phytonutrients help to enhance immunity and be implicated in the control of COVID-19. However, such measures should not replace social distancing, quarantine and special care.

Atomic structure of, and valine binding to the regulatory ACT domain of the Mycobacterium tuberculosis Rel protein.

The stringent response, regulated by the bifunctional (p)ppGpp synthetase/hydrolase Rel in mycobacteria, is critical for long-term survival of the drug-tolerant dormant state of Mycobacterium tuberculosis. During amino acid starvation, MtRel senses a drop in amino acid concentration and synthesizes the messengers pppGpp and ppGpp, collectively called (p)ppGpp. Here, we investigate the role of the regulatory 'Aspartokinase, Chorismate mutase and TyrA' (ACT) domain in MtRel. Using NMR spectroscopy approaches, we report the high-resolution structure of dimeric MtRel ACT which selectively binds to valine out of all other branched-chain amino acids tested. A set of MtRel ACT mutants were generated to identify the residues required for maintaining the head-to-tail dimer. Through NMR titrations, we determined the crucial residues for binding of valine and show structural rearrangement of the MtRel ACT dimer in the presence of valine. This study suggests the direct involvement of amino acids in (p)ppGpp accumulation mediated by MtRel independent to interactions with stalled ribosomes. Database Structural data are available in the PDB database under the accession number 6LXG.

Crystallographic and solution structure of the N-terminal domain of the Rel protein from Mycobacterium tuberculosis.

Modulation of intracellular guanosine 3',5'-bispyrophosphate ((p)ppGpp) level, the effector of the stringent response, is crucial for survival as well as optimal growth of prokaryotes and, thus, for bacterial pathogenesis and dormancy. In Mycobacterium tuberculosis (Mtb), (p)ppGpp synthesis and degradation are carried out by the bifunctional enzyme MtRel, which consists of 738 residues, including an N-terminal hydrolase- and synthetase-domain (N-terminal domain or NTD) and a C-terminus with a ribosome-binding site. Here, we present the first crystallographic structure of the enzymatically active MtRel NTD determined at 3.7 Å resolution. The structure provides insights into the residues of MtRel NTD responsible for nucleotide binding. Small-angle X-ray scattering experiments were performed to investigate the dimeric state of the MtRel NTD and possible substrate-dependent structural alterations.