Highlights d Structure of SARS-CoV-2 replication-transcription complex (RTC) with nsp13 helicases d The nsp13 NTPase domains sit in front of the RCT, constraining functional models d Nsp13 may drive RTC backtracking, affecting proofreading and template switching d Structural analysis of ADP-Mg 2+-bound NiRAN domain, a potential antiviral target
Lysine-specific demethylase 1 (LSD1) downregulates eukaryotic gene activity by demethylating mono and dimethylated Lys4 in histone H3. Elucidating the biochemical crosstalk of LSD1 with histone post-translational modifications (PTMs) is essential for developing LSD1-targeted therapeutics in human cancers. We interrogated the small ubiquitin-like modifier (SUMO) driven regulation of LSD1 activity with semisynthetic nucleosomes containing site-specifically methylated and sumoylated histones. We discovered that nucleosomes containing sumoylated histone H4 (suH4), a modification associated with gene repression, stimulate LSD1 activity by a mechanism dependent upon the SUMO-interaction motif in CoREST. Furthermore, the stimulatory effect of suH4 was spatially limited and did not extend to the demethylation of adjacent non-sumoylated nucleosomes. Thus, we have identified histone modification by SUMO as the first PTM that stimulates intranucleosomal demethylation by the developmentally critical LSD1-CoREST complex.
SUMMARYSARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated-transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryo-electron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template-product in complex with two molecules of the nsp13 helicase. The Nidovirus-order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12-thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapeutic development.
The C-terminal selective electrophilic activation of polypeptides is essential for site-specific peptide modification and conjugation techniques such as Native Chemical Ligation (NCL). Peptide C-terminal α-thioesters are particularly valuable precursors for NCL, due to their hydrolytic stability in aqueous buffers and reactivity toward thiol nucleophiles. The synthesis of peptide α-thioesters, however, requires harsh acidic conditions or complex chemical manipulations, which ultimately limits their functional group compatibility and broad utility. Herein, we report a readily accessible N-mercaptoethoxyglycinamide (MEGA) solid-phase linker for the facile synthesis of latent peptide α-thioesters. Incubating peptide-MEGA sequences with 2-mercaptoethanesulfonic acid at mildly acidic pH yielded α-thioesters that were directly used in NCL without purification. The MEGA linker yielded robust access to peptide α-thioesters ranging in length from 4 to 35 amino acids, and greatly simplified the synthesis of cyclic peptides. Finally, the high utility of MEGA was demonstrated by the one-pot synthesis of a functional analog of the Sunflower Trypsin Inhibitor 1.
The superfamily-1 helicase non-structural protein 13 (nsp13) is required for SARS-CoV-2 replication, making it an important antiviral therapeutic target. The mechanism and regulation of nsp13 has not been explored at the single-molecule level. Specifically, force-dependent unwinding experiments have yet to be performed for any coronavirus helicase. Here, using optical tweezers, we find that nsp13 unwinding frequency, processivity, and velocity increase substantially when a destabilizing force is applied to the dsRNA, suggesting a passive unwinding mechanism. These results, along with bulk assays, depict nsp13 as an intrinsically weak helicase that can be potently activated by picoNewton forces. Such force-dependent behavior contrasts the known behavior of other viral monomeric helicases, drawing stronger parallels to ring-shaped helicases. Our findings suggest that mechanoregulation, which may be provided by a directly bound RNA-dependent RNA polymerase, enables on-demand helicase activity on the relevant polynucleotide substrate during viral replication.
The post-translational modification of histones by the small ubiquitin-like modifier (SUMO) protein has been associated with gene regulation, centromeric localization, and double-strand break repair in eukaryotes. Although sumoylation of histone H4 was specifically associated with gene repression, this could not be proven due to the challenge of site-specifically sumoylating H4 in cells. Biochemical crosstalk between SUMO and other histone modifications, such as H4 acetylation and H3 methylation, that are associated with active genes also remains unclear. We addressed these challenges in mechanistic studies using an H4 chemically modified at Lys12 by SUMO-3 (H4K12su) and incorporated into mononucleosomes and chromatinized plasmids for functional studies. Mononucleosome-based assays revealed that H4K12su inhibits transcription-activating H4 tail acetylation by the histone acetyltransferase p300, as well as transcription-associated H3K4 methylation by the extended catalytic module of the Set1/COMPASS (complex of proteins associated with Set1) histone methyltransferase complex. Activator- and p300-dependent in vitro transcription assays with chromatinized plasmids revealed that H4K12su inhibits both H4 tail acetylation and RNA polymerase II-mediated transcription. Finally, cell-based assays with a SUMO-H4 fusion that mimics H4 tail sumoylation confirmed the negative crosstalk between histone sumoylation and acetylation/methylation. Thus, our studies establish the key role for histone sumoylation in gene silencing and its negative biochemical crosstalk with active transcription-associated marks in human cells.
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