The evolution of recognition specificities by the immune system depends on the generation of receptor diversity and on connecting the binding of new antigens with the initiation of downstream signaling. In plant immunity, the innate Nucleotide-Binding Leucine-Rich Repeat (NLR) receptor family enables antigen binding and immune signaling. In this study, we surveyed the NLR complements of 62 ecotypes of Arabidopsis thaliana and 54 lines of Brachypodium distachyon and identified a limited number of NLR subfamilies that show high allelic diversity. We show that the predicted specificity-determining residues cluster on the surfaces of Leucine-Rich Repeat domains, but the locations of the clusters vary among NLR subfamilies. By comparing NLR phylogeny, allelic diversity, and known functions of the Arabidopsis NLRs, we formulate a hypothesis for the emergence of direct and indirect pathogen-sensing receptors and of the autoimmune NLRs. These findings reveal the recurring patterns of evolution of innate immunity and can inform NLR engineering efforts.

RNA-directed transposon silencing operates in the mammalian soma and germline to safeguard genomic integrity. The piRNA pathway and the HUSH complex identify active transposons through recognition of their nascent transcripts, but mechanistic understanding of how these distinct pathways evolved is lacking. TASOR is an essential component of the HUSH complex. TASOR's DUF3715 domain adopts a pseudo-PARP structure and is required for transposon silencing in a manner independent of complex assembly. TEX15, an essential piRNA pathway factor, also contains the DUF3715 domain. Here, we show that TASOR's and TEX15's DUF3715 domain share extensive structural homology. We found that the DUF3715 domain arose in early eukaryotes and that in vertebrates it is restricted to TEX15, TASOR, and TASORB orthologues. While TASOR-like proteins are found throughout metazoa, TEX15 is vertebrate specific. The branching of TEX15 and TASOR-like DUF3715 domain likely occurred in early metazoan evolution. Remarkably, despite this vast evolutionary distance, the DUF3715 domain from divergent TEX15 sequences can functionally substitute for the same domain in TASOR and mediates transposon silencing. We have thus termed this domain of unknown function as the RNA-directed pseudo-PARP transposon silencing (RDTS) domain. In summary, we show an unexpected functional link between these critical transposon silencing pathways.

Monitoring the environment with serine/threonine protein kinases is critical for growth and survival of Mycobacterium tuberculosis, a devastating human pathogen. Protein kinase B (PknB) is a transmembrane serine/threonine protein kinase that acts as an essential regulator of mycobacterial growth and division. The PknB extracellular domain (ECD) consists of four repeats homologous to penicillin-binding protein and serine/threonine kinase associated (PASTA) domains, and binds fragments of peptidoglycan. These properties suggest that PknB activity is modulated by ECD binding to peptidoglycan substructures, however, the molecular mechanisms underpinning PknB regulation remain unclear. In this study, we report structural and genetic characterization of the PknB ECD. We determined the crystal structures of overlapping ECD fragments at near atomic resolution, built a model of the full ECD, and discovered a region on the C-terminal PASTA domain that has the properties of a ligand-binding site. Hydrophobic interaction between this surface and a bound molecule of citrate was observed in a crystal structure. Our genetic analyses in M. tuberculosis showed that nonfunctional alleles were produced either by deletion of any of single PASTA domain or by mutation of individual conserved residues lining the putative ligand-binding surface of the C-terminal PASTA repeat. These results define two distinct structural features necessary for PknB signal transduction, a fully extended ECD and a conserved, membrane-distal putative ligand-binding site.

Terpenes make up the largest class of natural products, with extensive chemical and structural diversity. Diterpenes, mostly isolated from plants and rarely prokaryotes, exhibit a variety of important biological activities and valuable applications, including providing antitumor and antibiotic pharmaceuticals. These natural products are constructed by terpene synthases, a class of enzymes that catalyze one of the most complex chemical reactions in biology: converting simple acyclic oligo-isoprenyl diphosphate substrates to complex polycyclic products via carbocation intermediates. Here we obtained the second ever crystal structure of a class II diterpene synthase from bacteria, tuberculosinol pyrophosphate synthase (i.e., Halimadienyl diphosphate synthase, MtHPS, or Rv3377c) from Mycobacterium tuberculosis (Mtb). This enzyme transforms (E,E,E)-geranylgeranyl diphosphate into tuberculosinol pyrophosphate (Halimadienyl diphosphate). Rv3377c is part of the Mtb diterpene pathway along with Rv3378c, which converts tuberculosinol pyrophosphate to 1-tuberculosinyl adenosine (1-TbAd). This pathway was shown to exist only in virulent Mycobacterium species, but not in closely related avirulent species, and was proposed to be involved in phagolysosome maturation arrest. To gain further insight into the reaction pathway and the mechanistically relevant enzyme substrate binding orientation, electronic structure calculation and docking studies of reaction intermediates were carried out. Results reveal a plausible binding mode of the substrate that can provide the information to guide future drug design and anti-infective therapies of this biosynthetic pathway.

Transcription of integrated DNA from viruses or transposable elements is tightly regulated to prevent pathogenesis. The Human Silencing Hub (HUSH), composed of Periphilin, TASOR and MPP8, silences transcriptionally active viral and endogenous transgenes. HUSH recruits effectors that alter the epigenetic landscape and chromatin structure, but how HUSH recognizes target loci and represses their expression remains unclear. We identify the physicochemical properties of Periphilin necessary for HUSH assembly and silencing. A disordered N-terminal domain (NTD) and structured C-terminal domain are essential for silencing. A crystal structure of the Periphilin-TASOR core complex shows Periphilin forms α -helical homodimers, which each bind a single TASOR molecule. The NTD binds RNA non-specifically and forms insoluble aggregates through an arginine/tyrosine-rich sequence reminiscent of low-complexity regions from self-associating RNA-binding proteins. Residues required for TASOR binding and aggregation were required for HUSH-dependent silencing and genome-wide deposition of repressive mark H3K9me3. The NTD was functionally complemented by low-complexity regions from certain RNA-binding proteins and proteins that form condensates or fibrils. Our work suggests the associative properties of Periphilin promote HUSH aggregation on nascent transcripts.

Bacterial growth and cell division are coordinated with hydrolysis of the peptidoglycan (PG) layer of the cell wall, but the mechanisms of regulation of extracellular PG hydrolases are not well understood. Here we report the biochemical, structural, and genetic analysis of the Mycobacterium tuberculosis homolog of the transmembrane PG-hydrolase regulator, FtsX. The purified FtsX extracellular domain binds the PG peptidase Rv2190c/RipC N-terminal segment, causing a conformational change that activates the enzyme. Deletion of ftsEX and ripC caused similar phenotypes in Mycobacterium smegmatis, as expected for genes in a single pathway. The crystal structure of the FtsX extracellular domain reveals an unprecedented fold containing two lobes connected by a flexible hinge. Mutations in the hydrophobic cleft between the lobes reduce RipC binding in vitro and inhibit FtsX function in M. smegmatis. These studies suggest how FtsX recognizes RipC and support a model in which a conformational change in FtsX links the cell division apparatus with PG hydrolysis.

The HUSH complex represses retroviruses, transposons and genes to maintain the integrity of vertebrate genomes. HUSH regulates deposition of the epigenetic mark H3K9me3, but how its three core subunits - TASOR, MPP8 and Periphilin - contribute to assembly and targeting of the complex remains unknown. Here, we define the biochemical basis of HUSH assembly and find that its modular architecture resembles the yeast RNA-induced transcriptional silencing complex. TASOR, the central HUSH subunit, associates with RNA processing components. TASOR is required for H3K9me3 deposition over LINE-1 repeats and repetitive exons in transcribed genes. In the context of previous studies, this suggests that an RNA intermediate is important for HUSH activity. We dissect the TASOR and MPP8 domains necessary for transgene repression. Structure-function analyses reveal TASOR bears a catalytically-inactive PARP domain necessary for targeted H3K9me3 deposition. We conclude that TASOR is a multifunctional pseudo-PARP that directs HUSH assembly and epigenetic regulation of repetitive genomic targets.

Summary

The Human Silencing Hub (HUSH) complex epigenetically represses retroviruses, transposons and genes in vertebrates. HUSH therefore maintains genome integrity and is central in the interplay between intrinsic immunity, transposable elements and transcriptional regulation. Comprising three subunits – TASOR, MPP8 and Periphilin – HUSH regulates SETDB1-dependent deposition of the transcriptionally repressive epigenetic mark H3K9me3 and recruits MORC2 to modify local chromatin structure. However the mechanistic roles of each HUSH subunit remain undetermined. Here we show that TASOR lies at the heart of HUSH, providing a platform for assembling the other subunits. Targeted epigenomic profiling supports the model that TASOR binds and regulates H3K9me3 specifically over LINE-1 repeats and other repetitive exons in transcribed genes. We find TASOR associates with several components of the nuclear RNA processing machinery and its modular domain architecture bears striking similarities to that of Chp1, the central component of the yeast RNA-induced transcriptional silencing (RITS) complex. Together these observations suggest that an RNA intermediate may be important for HUSH activity. We identify the TASOR domains necessary for HUSH assembly and transgene repression. Structural and genomic analyses reveal that TASOR contains a poly-ADP ribose polymerase (PARP) domain dispensable for assembly and chromatin localization, but critical for epigenetic regulation of target elements. This domain contains a degenerated and obstructed active site and has hence lost catalytic activity. Together our data demonstrate that TASOR is a pseudo-PARP critical for HUSH complex assembly and H3K9me3 deposition over its genomic targets.

Transcription of integrated DNA from viruses or transposable elements is tightly regulated to prevent pathogenesis. The Human Silencing Hub (HUSH), composed of Periphilin, TASOR and MPP8, silences transcriptionally active viral and endogenous transgenes. HUSH recruits effectors that alter the epigenetic landscape and chromatin structure, but how HUSH recognizes target loci and represses their expression remains unclear. We identify the physicochemical properties of Periphilin necessary for HUSH assembly and silencing. A disordered N-terminal domain (NTD) and structured C-terminal domain are essential for silencing. A crystal structure of the Periphilin-TASOR minimal core complex shows Periphilin forms an α-helical homodimer, bound by a single TASOR molecule. The NTD forms insoluble aggregates through an arginine/tyrosine-rich sequence reminiscent of low-complexity regions from self-associating RNA-binding proteins. Residues required for TASOR binding and aggregation were required for HUSH-dependent silencing and genome-wide deposition of repressive mark H3K9me3. The NTD was functionally complemented by low-complexity regions from certain RNA-binding proteins and proteins that form condensates or fibrils. Our work suggests the associative properties of Periphilin promote HUSH aggregation at target loci.

Soybean lipoxygenase (SLO) has served as a prototype for understanding the molecular origin of enzymatic rate accelerations. The double mutant (DM) L546A/L754A is considered a dramatic outlier, due to the unprecedented size and near temperature-independence of its primary kinetic isotope effect, low catalytic efficiency, and elevated enthalpy of activation. To uncover the physical basis of these features, we herein apply three structural probes: hydrogen-deuterium exchange mass spectrometry, room-temperature X-ray crystallography and EPR spectroscopy on four SLO variants (wild-type (WT) enzyme, DM, and the two parental single mutants, L546A and L754A). DM is found to incorporate features of each parent, with the perturbation at position 546 predominantly influencing thermally activated motions that connect the active site to a protein-solvent interface, while mutation at position 754 disrupts the ligand field and solvation near the cofactor iron. However, the expanded active site in DM leads to more active site water molecules and their associated hydrogen bond network, and the individual features from L546A and L754A alone cannot explain the aggregate kinetic properties for DM. Using recently published QM/MM-derived ground-state SLO-substrate complexes for WT and DM, together with the thorough structural analyses presented herein, we propose that the impairment of DM is the combined result of a repositioning of the reactive carbon of linoleic acid substrate with regard to both the iron cofactor and a catalytically linked dynamic region of protein.