Biology subjects

Tagliabracci, V. S.

Publications and source records attributed to Tagliabracci, V. S..

11 recordsLinked to original sources

bioRxiv2026

A minimal kinase-phosphatase system and its lipid substrates self-organize into dynamic patterns

Competing lipid kinases and phosphatases are critical for organizing cellular membranes, but whether a minimal system can autonomously organize proteins and lipids into dynamic spatiotemporal patterns is unknown. Here, we report the in vitro reconstitution of the Legionella phosphatidylinositol (PI) 3-kinase MavQ and PI 3-phosphatase SidP. Together with their lipid substrates, PI and PI 3-phosphate (PI3P), these enzymes form a minimal self-organizing system that generates ATP-dependent spatiotemporal patterns, including traveling waves, on model membranes. These behaviors arise from MavQs cooperative membrane binding, SidPs phosphatase activity, and the continual interconversion and redistribution of PI and PI3P within a conserved membrane pool. A reaction-diffusion model reproduces the observed dynamics and predicts that lipid conservation prevents patterns from propagating across membrane discontinuities, which we verify experimentally. Together, these findings establish enzymatic modification of membrane lipids as a distinct molecular strategy for biological pattern formation.

biophysics↗

bioRxiv2026

TIR-like NADases act in bacterial immunity and the RNA vault

Across all domains of life, organisms exploit NAD+ metabolism as a central line of defense against invading pathogens. Here, we show that domain of unknown function 4062 (DUF4062) is a widespread family of TIR-like NADases that hydrolyze NAD+ to ADP-ribose and nicotinamide. In bacteria, DUF4062 homologs form a previously unrecognized antiphage defense system, which we name Swaro[z]yc, that assembles with the phage portal into a supramolecular NADase complex to induce abortive infection. In eukaryotes, DUF4062 is found in TEP1, which we demonstrate functions as an active NADase within the RNA vault, an enigmatic organelle-like structure. Single-particle cryo-electron microscopy reveals ADP-ribose bound within the shoulder of both reconstituted and human brain vaults, while cryo-electron tomography positions TEP1 along the central axis at the shoulder. Thus, TEP1, like bacterial Swaro[z]yc, functions by depleting NAD+, providing new insight into the long-standing mystery of vault function.

biochemistry↗

bioRxiv2025

ACAD10 encodes two orphan enzymes in the ether lipid biosynthetic and salvage pathways

Ether lipids play critical roles in membrane dynamics, antioxidant defense, and signaling. They comprise ~20% of mammalian phospholipids, and disruptions in their metabolism cause severe genetic disorders and are associated with neurodegenerative and metabolic diseases. Ether lipids are synthesized de novo from glycolytic intermediates or salvaged from the diet. While the products of these pathways are known, several key enzymes remain unidentified, including the 1-O-alkylglycerol kinase and the 1-O-alkyl-2-acetyl-sn-glycero-3-phosphate phosphatase. Here, we show that acyl-CoA dehydrogenase member 10 (ACAD10) catalyzes the phosphorylation of 1-O-alkylglycerols and the dephosphorylation of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphate. Worms and mice lacking ACAD10 have reduced ether lipid levels and cannot salvage dietary alkylglycerols. Furthermore, individuals from the Akimel O'odham (Pima) tribe carrying ACAD10 polymorphisms also show decreased plasma ether lipid levels. Collectively, our findings resolve two long-standing gaps in ether lipid biochemistry and reveal a mechanistic link between ether lipid metabolism and a population-associated risk factor for type 2 diabetes.

biochemistry↗

bioRxiv2025

Bacterial ubiquitin ligase engineered for small molecule and protein target identification

The Legionella SidE effectors ubiquitinate host proteins independently of the canonical E1-E2 cascade. Here we engineer the SidE ligases to develop a modular proximity ligation approach for the identification of targets of small molecules and proteins, which we call SidBait. We validate the method with known small molecule-protein interactions and use it to identify CaMKII as an off-target interactor of the breast cancer drug ribociclib. Structural analysis and activity assays confirm that ribociclib binds the CaMKII active site and inhibits its activity. We further customize SidBait to identify protein-protein interactions, including substrates for enzymes, and discover the F-actin capping protein (CapZ) as a target of the Legionella effector RavB during infection. Structural and biochemical studies indicate that RavB allosterically binds CapZ and decaps actin, thus functionally mimicking eukaryotic CapZ interacting proteins. Collectively, our results establish SidBait as a reliable tool for identifying targets of small molecules and proteins.

molecular biology↗

bioRxiv2025

Metagenomic selections reveal diverse antiphage defenses in human and environmental microbiomes

To prevent phage infection, bacteria have developed an arsenal of antiphage defense systems. Using functional metagenomic selections, we identified new examples of these systems from human fecal, human oral, and grassland soil microbiomes. Our antiphage selections in Escherichia coli revealed over 200 putative defenses from 14 diverse bacterial phyla, highlighting the broad phylogenetic interoperability of these systems. Many defense systems were unrecognizable based on sequence or predicted structure, so could only be identified via functional assays. In mechanistic studies, we show that some defense systems encode nucleases that only degrade covalently modified phage DNA, but which accommodate diverse chemical modifications. We also identify outer membrane proteins that prevent phage adsorption and a set of previously unknown defense systems with diverse antiphage profiles and modalities. Most defenses acted against at least two phages, indicating that broadly acting systems are widely distributed among non-model bacteria.

microbiology↗

bioRxiv2024

Covalent inhibition of the SARS-CoV-2 NiRAN domain via an active-site cysteine

The kinase-like NiRAN domain of nsp12 in SARS-CoV-2 catalyzes the formation of the 5 RNA cap structure. This activity is required for viral replication, offering a new target for the development of antivirals. Here, we develop a high-throughput assay to screen for small molecule inhibitors targeting the SARS-CoV-2 NiRAN domain. We identified NCI-2, a compound with a reactive chloromethyl group that covalently binds to an active site cysteine (Cys53) in the NiRAN domain, inhibiting its activity. NCI-2 can enter cells, bind to, and inactivate ectopically expressed nsp12. A cryo-EM reconstruction of the SARS-CoV-2 replication-transcription complex (RTC) bound to NCI-2 offers a detailed structural blueprint for rational drug design. Although NCI-2 showed limited potency against SARS-CoV-2 replication in cells, our work lays the groundwork for developing more potent and selective inhibitors targeting the NiRAN domain. This approach presents a promising therapeutic strategy for effectively combating COVID-19 and potentially mitigating future coronavirus outbreaks.

biochemistry↗

bioRxiv2024

Biochemical and structural insights into a 5' to 3' RNA ligase reveal a potential role in tRNA ligation

ATP-grasp superfamily enzymes contain a hand-like ATP-binding fold and catalyze a variety of reactions using a similar catalytic mechanism. More than 30 protein families are categorized in this superfamily, and they are involved in a plethora of cellular processes and human diseases. Here we identify C12orf29 as an atypical ATP-grasp enzyme that ligates RNA. Human C12orf29 and its homologs auto-adenylate on an active site Lys residue as part of a reaction intermediate that specifically ligates RNA halves containing a 5-phosphate and a 3-hydroxyl. C12orf29 binds tRNA in cells and can ligate tRNA within the anticodon loop in vitro. Genetic depletion of c12orf29 in female mice alters global tRNA levels in brain. Furthermore, crystal structures of a C12orf29 homolog from Yasminevirus bound to nucleotides reveal a minimal and atypical RNA ligase fold with a unique active site architecture that participates in catalysis. Collectively, our results identify C12orf29 as an RNA ligase and suggest its involvement in tRNA biology. Significance StatementATP-grasp enzymes share an atypical ATP-binding fold and catalyze a diverse set of reactions involved in many essential cellular processes. We identified C12orf29 as an atypical ATP-grasp enzyme. Our biochemical and structural characterizations reveal this enzyme to be a 5 to 3 RNA ligase, structurally and functionally similar to the phage T4 RNA ligase. C12orf29 can ligate tRNAs in vitro and C12orf29 knockout female mice have altered tRNA levels in brain. We also report structures of C12orf29, which have revealed critical insights into the mode of ATP binding and catalysis. Our work suggests that C12orf29 may be a new player in the regulation of tRNAs.

biochemistry↗

bioRxiv2022

The mechanism of RNA capping by SARS-CoV-2

The SARS-CoV-2 RNA genome contains a 5-cap that facilitates translation of viral proteins, protection from exonucleases and evasion of the host immune response1-4. How this cap is made is not completely understood. Here, we reconstitute the SARS-CoV-2 7MeGpppA2-O-Me-RNA cap using virally encoded non-structural proteins (nsps). We show that the kinase-like NiRAN domain5 of nsp12 transfers RNA to the amino terminus of nsp9, forming a covalent RNA-protein intermediate (a process termed RNAylation). Subsequently, the NiRAN domain transfers RNA to GDP, forming the cap core structure GpppA-RNA. The nsp146 and nsp167 methyltransferases then add methyl groups to form functional cap structures. Structural analyses of the replication-transcription complex bound to nsp9 identified key interactions that mediate the capping reaction. Furthermore, we demonstrate in a reverse genetics system8 that the N-terminus of nsp9 and the kinase-like active site residues in the NiRAN domain are required for successful SARS-CoV-2 replication. Collectively, our results reveal an unconventional mechanism by which SARS-CoV-2 caps its RNA genome, thus exposing a new target in the development of antivirals to treat COVID-19.

biochemistry↗

bioRxiv2021

Structural and mechanistic basis for protein glutamylation by the kinase fold

The kinase domain transfers phosphate from ATP to substrates. However, the Legionella effector SidJ adopts a kinase fold yet catalyzes calmodulin (CaM)-dependent glutamylation to inactivate the SidE ubiquitin ligases. The structural and mechanistic basis in which the kinase domain catalyzes protein glutamylation is unknown. Here we present cryo-EM reconstructions of SidJ:CaM:SidE reaction intermediate complexes. We show that the kinase-like active site of SidJ adenylates an active site Glu in SidE resulting in the formation of a stable reaction intermediate complex. An insertion in the catalytic loop of the kinase domain positions the donor Glu near the acyl-adenylate for peptide bond formation. Our structural analysis led us to discover that the SidJ paralog SdjA is a glutamylase that differentially regulates the SidE-ligases during Legionella infection. Our results uncover the structural and mechanistic basis in which the kinase fold catalyzes non-ribosomal amino acid ligations and reveal an unappreciated level of SidE-family regulation.

biochemistry↗

bioRxiv2020

A Legionella effector ADP-ribosyltransferase inactivates glutamate dehydrogenase

ADP-ribosyltransferases (ARTs) are a widespread superfamily of enzymes frequently employed in pathogenic strategies of bacteria. Legionella pneumophila, the causative agent of Legionnaires disease, has acquired over 330 translocated effectors that showcase remarkable biochemical and structural diversity. Here we took a bioinformatic approach to search the Legionella effector repertoire for additional divergent members of the ART superfamily and identified an ART domain in Lpg0181. We show that L. pneumophila Lpg0181 targets a specific class of 120-kDa NAD+-dependent glutamate dehydrogenase (GDH) enzymes found in fungi and protists, including many natural hosts of Legionella. Lpg0181 targets a conserved arginine residue in the NAD+ -binding pocket of GDH, thereby blocking oxidative deamination of glutamate. While intracellular pathogens employ diverse virulence mechanisms to overcome host-limited nutrient availability, Lpg0181 is--to the best of our knowledge--the first example of a Legionella effector which directly targets a host metabolic enzyme.

biochemistry↗

bioRxiv2020

A Legionella effector kinase is activated by host inositol hexakisphosphate

The transfer of a phosphate from ATP to a protein substrate, a modification known as phosphorylation, is catalyzed by protein kinases. Protein kinases play a crucial role in virtually every cellular activity. Recent studies of atypical protein kinases have highlighted the structural similarity of the kinase superfamily despite notable differences in primary amino acid sequence. We searched for putative protein kinases in the intracellular bacterial pathogen, Legionella pneumophila and identified the Type-4 secretion system (T4SS) effector, Lpg2603 as a remote member of the protein kinase superfamily. We show that Lpg2603 is an active protein kinase with several atypical structural features. Importantly, we find that the eukaryotic-specific host signaling molecule, inositol hexakisphosphate (IP6) is required for Lpg2603 kinase activity. Crystal structures of Lpg2603 in the apo-form and bound to IP6 reveal active site rearrangement that allows for ATP binding and catalysis. Our results on the structure and activity of Lpg2603 reveal a unique mode of regulation of protein kinases and will aid future work into the function of this effector during Legionella pathogenesis.

biochemistry↗