A dual mechanism of action of AT-527 against SARS-CoV-2 polymerase.

The guanosine analog AT-527 represents a promising candidate against Severe Acute Respiratory Syndrome coronavirus type 2 (SARS-CoV-2). AT-527 recently entered phase III clinical trials for the treatment of COVID-19. Once in cells, AT-527 is converted into its triphosphate form, AT-9010, that presumably targets the viral RNA-dependent RNA polymerase (RdRp, nsp12), for incorporation into viral RNA. Here we report a 2.98 Å cryo-EM structure of the SARS-CoV-2 nsp12-nsp7-nsp82-RNA complex, showing AT-9010 bound at three sites of nsp12. In the RdRp active-site, one AT-9010 is incorporated at the 3' end of the RNA product strand. Its modified ribose group (2'-fluoro, 2'-methyl) prevents correct alignment of the incoming NTP, in this case a second AT-9010, causing immediate termination of RNA synthesis. The third AT-9010 is bound to the N-terminal domain of nsp12 - known as the NiRAN. In contrast to native NTPs, AT-9010 is in a flipped orientation in the active-site, with its guanine base unexpectedly occupying a previously unnoticed cavity. AT-9010 outcompetes all native nucleotides for NiRAN binding, inhibiting its nucleotidyltransferase activity. The dual mechanism of action of AT-527 at both RdRp and NiRAN active sites represents a promising research avenue against COVID-19.

Structural basis for the regulation of chemotaxis by MapZ in the presence of c-di-GMP.

The bacterial second messenger cyclic diguanylate monophosphate (c-di-GMP) mediates multiple aspects of bacterial physiology through binding to various effectors. In some cases, these effectors are single-domain proteins which only contain a PilZ domain. It remains largely unknown how single-domain PilZ proteins function and regulate their downstream targets. Recently, a single-domain PilZ protein, MapZ (PA4608), was identified to inhibit the activity of the methyltransferase CheR1. Here, crystal structures of the C-terminal domain of CheR1 containing SAH and of CheR1 in complex with c-di-GMP-bound MapZ are reported. It was observed that the binding site of MapZ in CheR1 partially overlaps with the SAH/SAM-binding pocket. Consequently, binding of MapZ blocks SAH/SAM binding. This provides direct structural evidence on the mechanism of inhibition of CheR1 by MapZ in the presence of c-di-GMP.

HDAC7 Ubiquitination by the E3 Ligase CBX4 Is Involved in Contextual Fear Conditioning Memory Formation.

Histone acetylation, an epigenetic modification, plays an important role in long-term memory formation. Recently, histone deacetylase (HDAC) inhibitors were demonstrated to promote memory formation, which raises the intriguing possibility that they may be used to rescue memory deficits. However, additional research is necessary to clarify the roles of individual HDACs in memory. In this study, we demonstrated that HDAC7, within the dorsal hippocampus of C57BL6J mice, had a late and persistent decrease after contextual fear conditioning (CFC) training (4-24 h), which was involved in long-term CFC memory formation. We also showed that HDAC7 decreased via ubiquitin-dependent degradation. CBX4 was one of the HDAC7 E3 ligases involved in this process. Nur77, as one of the target genes of HDAC7, increased 6-24 h after CFC training and, accordingly, modulated the formation of CFC memory. Finally, HDAC7 was involved in the formation of other hippocampal-dependent memories, including the Morris water maze and object location test. The current findings facilitate an understanding of the molecular and cellular mechanisms of HDAC7 in the regulation of hippocampal-dependent memory.SIGNIFICANCE STATEMENT The current findings demonstrated the effects of histone deacetylase 7 (HDAC7) on hippocampal-dependent memories. Moreover, we determined the mechanism of decreased HDAC7 in contextual fear conditioning (CFC) through ubiquitin-dependent protein degradation. We also verified that CBX4 was one of the HDAC7 E3 ligases. Finally, we demonstrated that Nur77, as one of the important targets for HDAC7, was involved in CFC memory formation. All of these proteins, including HDAC7, CBX4, and Nur77, could be potential therapeutic targets for preventing memory deficits in aging and neurological diseases.