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, inhibiting 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

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 Pro-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 Å. 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