ABSTRACT
PIWI proteins use PIWI-interacting RNAs (piRNAs) to identify and silence transposable elements and thereby maintain genome integrity between metazoan generations1. The targeting of transposable elements by PIWI has been compared to mRNA target recognition by Argonaute proteins2,3, which use microRNA (miRNA) guides, but the extent to which piRNAs resemble miRNAs is not known. Here we present cryo-electron microscopy structures of a PIWI-piRNA complex from the sponge Ephydatia fluviatilis with and without target RNAs, and a biochemical analysis of target recognition. Mirroring Argonaute, PIWI identifies targets using the piRNA seed region. However, PIWI creates a much weaker seed so that stable target association requires further piRNA-target pairing, making piRNAs less promiscuous than miRNAs. Beyond the seed, the structure of PIWI facilitates piRNA-target pairing in a manner that is tolerant of mismatches, leading to long-lived PIWI-piRNA-target interactions that may accumulate on transposable-element transcripts. PIWI ensures targeting fidelity by physically blocking the propagation of piRNA-target interactions in the absence of faithful seed pairing, and by requiring an extended piRNA-target duplex to reach an endonucleolytically active conformation. PIWI proteins thereby minimize off-targeting cellular mRNAs while defending against evolving genomic threats.
Subject(s)
Nucleic Acid Conformation , Porifera , RNA, Small Interfering/chemistry , RNA, Small Interfering/genetics , Animals , Argonaute Proteins/chemistry , Argonaute Proteins/metabolism , Argonaute Proteins/ultrastructure , Cryoelectron Microscopy , Models, Molecular , Porifera/genetics , Porifera/metabolism , Porifera/ultrastructure , RNA, Small Interfering/metabolism , RNA, Small Interfering/ultrastructure , Substrate SpecificityABSTRACT
CDC7 is an essential Ser/Thr kinase that acts upon the replicative helicase throughout the S phase of the cell cycle and is activated by DBF4. Here, we present crystal structures of a highly active human CDC7-DBF4 construct. The structures reveal a zinc-finger domain at the end of the kinase insert 2 that pins the CDC7 activation loop to motif M of DBF4 and the C lobe of CDC7. These interactions lead to ordering of the substrate-binding platform and full opening of the kinase active site. In a co-crystal structure with a mimic of MCM2 Ser40 phosphorylation target, the invariant CDC7 residues Arg373 and Arg380 engage phospho-Ser41 at substrate P+1 position, explaining the selectivity of the S-phase kinase for Ser/Thr residues followed by a pre-phosphorylated or an acidic residue. Our results clarify the role of DBF4 in activation of CDC7 and elucidate the structural basis for recognition of its preferred substrates.
Subject(s)
Cell Cycle Proteins/chemistry , Protein Serine-Threonine Kinases/chemistry , Catalytic Domain , Cell Cycle Proteins/metabolism , Humans , Molecular Docking Simulation , Phosphorylation , Protein Binding , Protein Serine-Threonine Kinases/metabolism , Substrate Specificity , Zinc FingersABSTRACT
Psammaplin A (11c) is a marine metabolite previously reported to be a potent inhibitor of two classes of epigenetic enzymes: histone deacetylases and DNA methyltransferases. The design and synthesis of a focused library based on the psammaplin A core has been carried out to probe the molecular features of this molecule responsible for its activity. By direct in vitro assay of the free thiol generated upon reduction of the dimeric psammaplin scaffold, we have unambiguously demonstrated that 11c functions as a natural prodrug, with the reduced form being highly potent against HDAC1 in vitro (IC(50) 0.9 nM). Furthermore, we have shown it to have high isoform selectivity, being 360-fold selective for HDAC1 over HDAC6 and more than 1000-fold less potent against HDAC7 and HDAC8. SAR around our focused library revealed a number of features, most notably the oxime functionality to be important to this selectivity. Many of the compounds show significant cytotoxicity in A549, MCF7, and W138 cells, with the SAR of cytotoxicity correlating to HDAC inhibition. Furthermore, compound treatment causes upregulation of histone acetylation but little effect on tubulin acetylation. Finally, we have found no evidence for 11c functioning as a DNMT inhibitor.