Biology subjects

Finocchio, G.

Publications and source records attributed to Finocchio, G..

4 recordsLinked to original sources

bioRxiv2023

RNA-guided RNA silencing by an Asgard archaeal Argonaute

Eukaryotic Argonaute proteins achieve gene repression and defense against viruses and transposons by RNA-guided RNA silencing. By contrast, known prokaryotic Argonautes adopt single-stranded DNA as guides and/or targets, leaving the evolutionary origin of RNA-guided RNA silencing elusive. Here, we show an evolutionary expansion of Asgard archaeal Argonautes (asAgos), including the discovery of HrAgo1 from the Lokiarchaeon Candidatus Harpocratesius repetitus that shares a common origin with eukaryotic PIWI proteins. HrAgo1 exhibits RNA-guided RNA cleavage in vitro and RNA silencing in human cells. The cryo-EM structure of HrAgo1 combined with quantitative single-molecule experiments reveals that HrAgo1 possesses hybrid structural features and target binding modes bridging those of the eukaryotic AGO and PIWI clades. Finally, genomic evidence suggests that eukaryotic Dicer-like processing of double-stranded RNA likely emerged as a mechanism of generating guide RNA for asAgos prior to eukaryogenesis. Our study provides new insights into the evolutionary origin and plasticity of Argonaute-based RNA silencing.

microbiology↗

bioRxiv2023

Target DNA-dependent activation mechanism of the prokaryotic immune system SPARTA

In both prokaryotic and eukaryotic innate immune systems, TIR domains function as NADases that degrade the key metabolite NAD+ or generate signaling molecules. Catalytic activation of TIR domains requires oligomerization, but how this is achieved varies in distinct immune systems. In the Short prokaryotic Argonaute (pAgo)/TIR-APAZ (SPARTA) immune system, TIR NADase activity is triggered upon guide RNA-mediated recognition of invading DNA by an unknown mechanism. Here, we describe cryo-EM structures of SPARTA in the inactive monomeric and target DNA-activated tetrameric states. The monomeric SPARTA structure reveals that in the absence of target DNA, a C-terminal tail of TIR-APAZ occupies the nucleic acid binding cleft formed by the pAgo and TIR-APAZ subunits, suppressing SPARTA activation. In the active tetrameric SPARTA complex, guide RNA-mediated target DNA binding displaces the C-terminal tail and induces conformational changes in pAgo that facilitate SPARTA-SPARTA dimerization. Concurrent release and rotation of one TIR domain allow it to form a composite NADase catalytic site with the other TIR domain within the dimer, and generate a self-complementary interface that mediates cooperative tetramerization. Combined, this study provides critical insights into the structural architecture of SPARTA and the molecular mechanism underlying target DNA-dependent oligomerization and catalytic activation. KEY POINTSO_LIInactive monomeric SPARTA is autoinhibited by the C-terminal tail of TIR-APAZ C_LIO_LITarget DNA recognition causes C-terminal tail release, pAgo restructuring, and dimerization C_LIO_LITIR domain rotation enables catalytic activation and cooperative tetramer formation C_LI

biochemistry↗

bioRxiv2023

Plasticity of the binding pocket in peptide transporters underpins promiscuous substrate recognition

Proton-coupled oligopeptide transporters (POTs) are promiscuous transporters of the Major Facilitator Superfamily, that constitute the main route of entry for a wide range of dietary peptides and orally administrated peptidomimetic drugs. Given their clinical and pathophysiological relevance, several bacterial and mammalian POT homologs have been extensively studied on a structural and molecular level. However, the molecular basis of recognition and transport of the wide range of peptide substrates has remained elusive. Here we present 14 X-ray structures of the bacterial POT DtpB in complex with chemically diverse di- and tripeptides, providing novel insights into the plasticity of the conserved central binding cavity. We analyzed binding affinities for more than 80 peptides and monitored uptake by a fluorescence-based transport assay. To probe if all natural 8400 di- and tripeptides can bind to DtpB, we employed state-of-the-art molecular docking and machine learning and conclude that peptides of a specific subset with compact hydrophobic residues are the best DtpB binders.

biophysics↗

bioRxiv2022

Cryo-EM structure of an atypical proton-coupled peptide transporter: Di- and tripeptide permease C

Proton-coupled Oligopeptide Transporters (POTs) of the Major Facilitator Superfamily (MFS) mediate the uptake of short di- and tripeptides in all phyla of life. POTs are thought to constitute the most promiscuous class of MFS transporters, with the potential to transport more than 8400 unique substrates. Over the past two decades, transport assays and biophysical studies have shown that various orthologues and paralogues display differences in substrate selectivity. The E. coli genome codes for four different POTs, known as Di- and Tripeptide permeases A-D (DtpA-D). DtpC was shown previously to favor positively charged peptides as substrates. In this study, we describe, how we determined the structure of the 53 kDa DtpC by cryogenic electron microscopy (cryo-EM), and provide structural insights into the ligand specificity of this atypical POT. We collected and analyzed data on the transporter fused to split superfolder GFP (split sfGFP), in complex with a 52 kDa macrobody and with a 13 kDa nanobody. The latter sample was more stable, rigid and a significant fraction dimeric, allowing us to reconstruct a 3D volume of DtpC at a resolution of 2.7 [A]. This work provides a molecular explanation for the selectivity of DtpC, and highlights the value of small and rigid fiducial markers such as nanobodies for structure determination of low molecular weight integral membrane proteins lacking soluble domains.

biophysics↗