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At least 1,027 records · Page 57
All sourcesNASA NTRSDOE OSTI
NASA NTRS1999

The Effect of Spaceflight on Bone Cell Cultures

Understanding the response of bone to mechanical loading (unloading) is extremely important in defining the means of adaptation of the body to a variety of environmental conditions such as during heightened physical activity or in extended explorations of space or the sea floor. The mechanisms of the adaptive response of bone are not well defined, but undoubtedly they involve changes occurring at the cellular level of bone structure. This proposal has intended to examine the hypothesis that the loading (unloading) response of bone is mediated by specific cells through modifications of their activity cytoskeletal elements, and/or elaboration of their extracellular matrices. For this purpose, this laboratory has utilized the results of a number of previous studies defining molecular biological, biochemical, morphological, and ultrastructural events of the reproducible mineralization of a primary bone cell (osteoblast) culture system under normal loading (1G gravity level). These data and the culture system then were examined following the use of the cultures in two NASA shuttle flights, STS-59 and STS-63. The cells collected from each of the flights were compared to respective synchronous ground (1G) control cells examined as the flight samples were simultaneously analyzed and to other control cells maintained at 1G until the time of shuttle launch, at which point they were terminated and studied (defined as basal cells). Each of the cell cultures was assayed in terms of metabolic markers- gene expression; synthesis and secretion of collagen and non-collagenous proteins, including certain cytoskeletal components; assembly of collagen into macrostructural arrays- formation of mineral; and interaction of collagen and mineral crystals during calcification of the cultures. The work has utilized a combination of biochemical techniques (radiolabeling, electrophoresis, fluorography, Western and Northern Blotting, and light microscopic immunofluorescence) and structural methods (conventional and high voltage electron microscopy, inununocytochemistry, stereomicroscopy, and 3D image reconstruction). The studies have provided new knowledge of aspects of bone cell development and structural regulation, extracellular matrix assembly, and mineralization during spaceflight and under normal gravity. The information has contributed to insights into the means in general by which cells respond and adapt to different conditions of gravity (loading). The data may as well have suggested an underlying basis for the observed loss of bone by vertebrates, including man, in microgravity; and these scientific results may have implications for understanding bone loss following fracture healing and extended periods of inactivity such as during long-term bedrest.

Landis, William J.↗
DOE OSTI2021

A synthetic promoter system for well-controlled protein expression with different carbon sources in Saccharomyces cerevisiae

Saccharomyces cerevisiae is an important synthetic biology chassis for microbial production of valuable molecules. Promoter engineering has been frequently applied to generate more synthetic promoters with a variety of defined characteristics in order to achieve a well-regulated genetic network for high production efficiency. Galactose-inducible (GAL) expression systems, composed of GAL promoters and multiple GAL regulators, have been widely used for protein overexpression and pathway construction in S. cerevisiae. However, the function of each element in synthetic promoters and how they interact with GAL regulators are not well known. Here, a library of synthetic GAL promoters demonstrate that upstream activating sequences (UASs) and core promoters have a synergistic relationship that determines the performance of each promoter under different carbon sources. We found that the strengths of synthetic GAL promoters could be fine-tuned by manipulating the sequence, number, and substitution of UASs. Core promoter replacement generated synthetic promoters with a twofold strength improvement compared with the GAL1 promoter under multiple different carbon sources in a strain with GAL1 and GAL80 engineering. These results represent an expansion of the classic GAL expression system with an increased dynamic range and a good tolerance of different carbon sources. In this study, the effect of each element on synthetic GAL promoters has been evaluated and a series of well-controlled synthetic promoters are constructed. By studying the interaction of synthetic promoters and GAL regulators, synthetic promoters with an increased dynamic range under different carbon sources are created.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2004

Modeled microgravity-induced protein kinase C isoform expression in human lymphocytes

In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited in both microgravity and modeled microgravity (MMG) as reflected by diminished DNA synthesis in peripheral blood lymphocytes and their locomotion through gelled type I collagen. Direct activation of protein kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas the calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 1 g and MMG culture. Human lymphocytes were cultured and harvested at 24, 48, 72, and 96 h, and serial samples were assessed for locomotion by using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta, and -epsilon was assessed by RT-PCR, flow cytometry, and immunoblotting. Results indicated that PKC isoforms delta and epsilon were downregulated by >50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 1-g controls. Events upstream of PKC, such as phosphorylation of phospholipase Cgamma in MMG, revealed accumulation of inactive enzyme. Depressed calcium-independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than PKC, but after ligand-receptor interaction.

Lymphocytes/enzymology↗
NASA NTRS1996

Sequence, overproduction and purification of Vibrio proteolyticus ribosomal protein L18 for in vitro and in vivo studies

A strategy suggested by comparative genomic studies was used to amplify the entire Vibrio proteolyticus (Vp) gene for ribosomal protein L18. Vp L18 and its flanking regions were sequenced and compared with the deduced amino acid (aa) sequences of other known L18 proteins. A 26-aa residue segment at the carboxy terminus contains many strongly conserved residues and may be critical for the L18 interaction with 5S rRNA. This approach should allow rapid characterization of L18 from large numbers of bacteria. Both Vp L18 and Escherichia coli (Ec) L18 were overproduced and purified using a T7 expression vector which fuses an N-terminal peptide segment (His-tag) containing 6 histidine residues to the recombinant protein. The purified fusion proteins, Vp His::L18 and Ec His::L18, were both found to bind to either the Vp 5S or Ec 5S rRNAs in vitro. Vp His::L18 protein was also shown to incorporate into Ec ribosomes in vivo. This His-tag strategy likely will have general applicability for the study of ribosomal proteins in vitro and in vivo.

Non-NASA Center↗
NASA NTRS1993

Detergent solubilization of the EGF receptor from A431 cells

Functional reconstitution of purified preparations of human epidermal growth factor receptor (EGFR) requires dissociation of the protein from its plasma membrane lipid environment. Solubilization of membrane proteins in this manner requires the use of detergents, which are known to disrupt plasma membrane lipid/protein interactions. We have investigated the ability of three nonionic detergents to solubilize the human EGFR selectively, and have also analyzed the effect of these various treatments on the intrinsic tyrosyl kinase activity of the receptor. The nonionic detergent known as n-octyl glucoside (n-octyl beta-D-glucopyranoside) was found to give the best combination of selectivity, yield, and maintenance of enzymatic activity of the human EGFR.

Non-NASA Center↗
DOE OSTI2021

Protein-Mimetic Self-Assembly with Synthetic Macromolecules

Protein-mimetic amphiphiles have significant promise as a platform to access the complex functions of natural biological materials and incorporate the tunability and environmental resilience of synthetic materials. The fields of polymer chemistry and chemical biology have concurrently approached the development of biomimetic amphiphiles with materials ranging from random amphiphilic copolymers to peptide–lipid conjugates. In this Perspective, we incorporate strategies from diverse chemical arenas for controlling assembled morphologies and dynamics of protein-mimetic synthetic macromolecules. An overview of significant advances in peptide amphiphiles and single-chain polymer nanoparticles provides the foundation for comparing recent advances in the implementation of multiple intermolecular interactions and computational strategies to fine-tune the assembled structures. We aim to bridge these fields, combining insights from multiple disciplines to inspire new approaches for the development of protein-mimetic materials, as these assemblies have far-reaching applications including in the development of new sensors, catalysts, and therapeutics.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Ca X ML: Chemistry‐informed machine learning explains mutual changes between protein conformations and calcium ions in calcium‐binding proteins using structural and topological features

Proteins' flexibility is a feature in communicating changes in cell signaling instigated by binding with secondary messengers, such as calcium ions, associated with the coordination of muscle contraction, neurotransmitter release, and gene expression. When binding with the disordered parts of a protein, calcium ions must balance their charge states with the shape of calcium-binding proteins and their versatile pool of partners depending on the circumstances they transmit. Accurately determining the ionic charges of those ions is essential for understanding their role in such processes. However, it is unclear whether the limited experimental data available can be effectively used to train models to accurately predict the charges of calcium-binding protein variants. Here, we developed a chemistry-informed, machine-learning algorithm that implements a game theoretic approach to explain the output of a machine-learning model without the prerequisite of an excessively large database for high-performance prediction of atomic charges. We used the ab initio electronic structure data representing calcium ions and the structures of the disordered segments of calcium-binding peptides with surrounding water molecules to train several explainable models. Network theory was used to extract the topological features of atomic interactions in the structurally complex data dictated by the coordination chemistry of a calcium ion, a potent indicator of its charge state in protein. Our design created a computational tool of Ca X ML, which provided a framework of explainable machine learning model to annotate ionic charges of calcium ions in calcium-binding proteins in response to the chemical changes in an environment. Our framework will provide new insights into protein design for engineering functionality based on the limited size of scientific data in a genome space.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

The crystal structure of bacteriophage λ RexA provides novel insights into the DNA binding properties of Rex-like phage exclusion proteins

Abstract RexA and RexB function as an exclusion system that prevents bacteriophage T4rII mutants from growing on Escherichia coli λ phage lysogens. Recent data established that RexA is a non-specific DNA binding protein that can act independently of RexB to bias the λ bistable switch toward the lytic state, preventing conversion back to lysogeny. The molecular interactions underlying these activities are unknown, owing in part to a dearth of structural information. Here, we present the 2.05-Å crystal structure of the λ RexA dimer, which reveals a two-domain architecture with unexpected structural homology to the recombination-associated protein RdgC. Modelling suggests that our structure adopts a closed conformation and would require significant domain rearrangements to facilitate DNA binding. Mutagenesis coupled with electromobility shift assays, limited proteolysis, and double electron–electron spin resonance spectroscopy support a DNA-dependent conformational change. In vivo phenotypes of RexA mutants suggest that DNA binding is not a strict requirement for phage exclusion but may directly contribute to modulation of the bistable switch. We further demonstrate that RexA homologs from other temperate phages also dimerize and bind DNA in vitro. Collectively, these findings advance our mechanistic understanding of Rex functions and provide new evolutionary insights into different aspects of phage biology.

Biochemistry & Molecular Biology↗
DOE OSTI2020

Molecular recognition of a host protein by NS1 of pandemic and seasonal influenza A viruses

The 1918 influenza A virus (IAV) caused the most severe flu pandemic in recorded human history. Nonstructural protein 1 (NS1) is an important virulence factor of the 1918 IAV. NS1 antagonizes host defense mechanisms through interactions with multiple host factors. One pathway by which NS1 increases virulence is through the activation of phosphoinositide 3-kinase (PI3K) by binding to its p85β subunit. Here we present the mechanism underlying the molecular recognition of the p85β subunit by 1918 NS1. Using X-ray crystallography, we determine the structure of 1918 NS1 complexed with p85β of human PI3K. We find that the 1918 NS1 effector domain (1918 NS1 ED ) undergoes a conformational change to bind p85β. Using NMR relaxation dispersion and molecular dynamics simulation, we identify that free 1918 NS1 ED exists in a dynamic equilibrium between p85β-binding–competent and –incompetent conformations in the submillisecond timescale. Moreover, we discover that NS1 ED proteins of 1918 (H1N1) and Udorn (H3N2) strains exhibit drastically different conformational dynamics and binding kinetics to p85β. These results provide evidence of strain-dependent conformational dynamics of NS1. Using kinetic modeling based on the experimental data, we demonstrate that 1918 NS1 ED can result in the faster hijacking of p85β compared to Ud NS1 ED , although the former has a lower affinity to p85β than the latter. Our results suggest that the difference in binding kinetics may impact the competition with cellular antiviral responses for the activation of PI3K. We anticipate that our findings will increase the understanding of the strain-dependent behaviors of influenza NS1 proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A Surface Exposed, Two-Domain Lipoprotein Cargo of a Type XI Secretion System Promotes Colonization of Host Intestinal Epithelia Expressing Glycans

The only known required component of the newly described Type XI secretion system (TXISS) is an outer membrane protein (OMP) of the DUF560 family. TXISS OMPs are broadly distributed across proteobacteria, but properties of the cargo proteins they secrete are largely unexplored. We report biophysical, histochemical, and phenotypic evidence that Xenorhabdus nematophila NilC is surface exposed. Biophysical data and structure predictions indicate that NilC is a two-domain protein with a C-terminal, 8-stranded β-barrel. This structure has been noted as a common feature of TXISS effectors and may be important for interactions with the TXISS OMP . The NilC N-terminal domain is more enigmatic, but our results indicate it is ordered and forms a β-sheet structure, and bioinformatics suggest structural similarities to carbohydrate-binding proteins. X. nematophila NilC and its presumptive TXISS OMP partner NilB are required for colonizing the anterior intestine of Steinernema carpocapsae nematodes: the receptacle of free-living, infective juveniles and the anterior intestinal cecum (AIC) in juveniles and adults. We show that, in adult nematodes, the AIC expresses a Wheat Germ Agglutinin (WGA)-reactive material, indicating the presence of N-acetylglucosamine or N-acetylneuraminic acid sugars on the AIC surface. A role for this material in colonization is supported by the fact that exogenous addition of WGA can inhibit AIC colonization by X. nematophila. Conversely, the addition of exogenous purified NilC increases the frequency with which X. nematophila is observed at the AIC, demonstrating that abundant extracellular NilC can enhance colonization. NilC may facilitate X. nematophila adherence to the nematode intestinal surface by binding to host glycans, it might support X. nematophila nutrition by cleaving sugars from the host surface, or it might help protect X. nematophila from nematode host immunity. Proteomic and metabolomic analyses of wild type X. nematophila compared to those lacking nilB and nilC revealed differences in cell wall and secreted polysaccharide metabolic pathways. Additionally, purified NilC is capable of binding peptidoglycan, suggesting that periplasmic NilC may interact with the bacterial cell wall. Overall, these findings support a model that NilB-regulated surface exposure of NilC mediates interactions between X. nematophila and host surface glycans during colonization. This is a previously unknown function for a TXISS.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

In-situ Investigations on Gold Nanoparticles Stabilization Mechanisms in Biological Environments Containing HSA

Nanoparticles (NPs) developments advance innovative biomedical applications. However, complex interactions and the low colloidal stability of NPs in biological media restrict their widespread utilization. The influence of NPs properties on the colloidal stability for gold NPs with 5 and 40 nm in diameter with two surface modifications, methoxy-polyethylene glycol-sulfhydryl (PEG) and citrate, in NaCl and human serum albumin (HSA) protein solution, is investigated. This study is based on small-angle X-ray scattering (SAXS) methods allowing the in-situ monitoring of interactions in physiological conditions. The PEG coating provides high colloidal stability for NPs of both sizes. For 5 nm NPs in NaCl solution, a stable 3D self-assembled body-centered cubic (BCC) arrangement is detected with an interparticle distance of 20.7 ± 0.1 nm. In protein solution, this distance increases to 21.9 ± 0.1 nm by protein penetration inside the ordered structure. For citrate-capped NPs, a different mechanism is observed. The protein particles attach to the NPs surfaces, and an appropriate concentration of proteins results in a stable suspension. Cryogenic transmission electron microscopy (Cryo-TEM), UV–visible spectroscopy, and dynamic light scattering (DLS) support the SAXS results. The findings will pave the way to design and synthesize NPs with controlled behaviors in biomedical applications.

36 MATERIALS SCIENCE↗
DOE OSTI2026

Exploring the binding properties and activities of ancestral expansins

Bacterial expansins are non-lytic proteins capable of loosening cellulose networks, offering promising applications in agriculture, biotechnology, and material science. Their ability to disrupt noncovalent interactions in biopolymer matrices such as cellulose and chitin positions them as valuable tools for upgrading abundant natural materials. However, their industrial use remains limited due to their relatively low wall-loosening activity compared to plant expansins. To address this limitation, we applied Ancestral Sequence Resurrection (ASR) to reconstruct and characterize ancient variants of the Bacillus subtilis expansin BsEXLX1. ASR is a powerful evolutionary tool that enables the inference and synthesis of ancestral proteins, allowing researchers to explore functional traits that may have been lost over time. This approach not only provides insights into protein evolution but also facilitates the design of proteins with enhanced properties, such as improved substrate affinity or structural stability. In this study, we combined biochemical and biophysical assays to evaluate the activity and binding behavior of ancestral expansins. Our results reveal that ancestral variants exhibit increased cellulose affinity, reduced binding to acidic polysaccharides, and greater salt resistance. Furthermore, these traits enhance their wall-loosening activity and demonstrate the utility of ASR in engineering surface-active proteins for industrial applications, particularly in biomass processing and cellulose modification.

09 BIOMASS FUELS↗
DOE OSTI2020

The plant endoplasmic reticulum: an organized chaos of tubules and sheets with multiple functions

The endoplasmic reticulum is a fascinating organelle at the core of the secretory pathway. It is responsible for the synthesis of one third of the cellular proteome and, in plant cells, it produces receptors and transporters of hormones as well as the proteins responsible for the biosynthesis of critical components of a cellulosic cell wall. The endoplasmic reticulum structure resembles a spider-web network of interconnected tubules and cisternae that pervades the cell. The study of the dynamics and interaction of this organelles with other cellular structures such as the plasma membrane, the Golgi apparatus and the cytoskeleton, have been permitted by the implementation of fluorescent protein and advanced confocal imaging. In this review,we report on the findings that contributed towards the understanding of the endoplasmic reticulum morphology and function with the aid of fluorescent proteins, focusing on the contributions provided by pioneering work from the lab of the late Professor Chris Hawes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

An Activity-Based Oxaziridine Platform for Identifying and Developing Covalent Ligands for Functional Allosteric Methionine Sites: Redox-Dependent Inhibition of Cyclin-Dependent Kinase 4

Activity-based protein profiling (ABPP) is a versatile strategy for identifying and characterizing functional protein sites and compounds for therapeutic development. However, the vast majority of ABPP methods for covalent drug discovery target highly nucleophilic amino acids such as cysteine or lysine. Here, we report a methionine-directed ABPP platform using Redox-Activated Chemical Tagging (ReACT), which leverages a biomimetic oxidative ligation strategy for selective methionine modification. Application of ReACT to oncoprotein cyclin-dependent kinase 4 (CDK4) as a representative high-value drug target identified three new ligandable methionine sites. We then synthesized a methionine-targeting covalent ligand library bearing a diverse array of heterocyclic, heteroatom, and stereochemically rich substituents. ABPP screening of this focused library identified 1oxF11 as a covalent modifier of CDK4 at an allosteric M169 site. This compound inhibited kinase activity in a dose-dependent manner on purified protein and in breast cancer cells. Further investigation of 1oxF11 found prominent cation-π and H-bonding interactions stabilizing the binding of this fragment at the M169 site. Quantitative mass-spectrometry studies validated 1oxF11 ligation of CDK4 in breast cancer cell lysates. Further biochemical analyses revealed cross-talk between M169 oxidation and T172 phosphorylation, where M169 oxidation prevented phosphorylation of the activating T172 site on CDK4 and blocked cell cycle progression. Finally, by identifying a new mechanism for allosteric methionine redox regulation on CDK4 and developing a unique modality for its therapeutic intervention, this work showcases a generalizable platform that provides a starting point for engaging in broader chemoproteomics and protein ligand discovery efforts to find and target previously undruggable methionine sites.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Unraveling Electronic and Vibrational Coherences Following a Charge Transfer Process in a Photosystem II Reaction Center

A reaction center is a unique biological system that performs the initial charge separation within a Photosystem II (PSII) multiunit enzyme, which eventually drives the catalytic water-splitting in plants and algae. The possible role of quantum coherences coinciding with the energy and charge transfer processes in PSII reaction center is one of the active areas of research. Here, we study these quantum coherences by using a numerically exact method on an excitonic dimer model, including linear vibronic coupling and employing optimal parameters from experimental two-dimensional coherent spectroscopic measurements. This enables us to precisely capture the excitonic interaction between pigments and the dissipation of the energy from electronic and charge-transfer (CT) states to the protein environment. We employ the time nonlocal (TNL) quantum master equation to calculate the population dynamics, which yields numerically reliable results. The calculated results show that, due to the strong dissipation, the lifetime of electronic coherence is too short to have direct participation in the charge transfer processes. However, there are long-lived vibrational coherences present in the system at frequencies close to the excitionic energy gap. These are strongly coupled with the electronic coherences, which makes the detection of the electronic coherences with conventional techniques very challenging. Additionally, we unravel the strong excitonic interaction of radical pair (PD1 and PD2) in the reaction center, which results in a long-lived electronic coherence of >100 fs, even at room temperature. Our work provide important physical insight to the charge separation process in PSII reaction center, which may be helpful for better understanding of photophysical processes in other natural and artificial light-harvesting systems.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗
DOE OSTI2020

Nop9 recognizes structured and single-stranded RNA elements of preribosomal RNA

Nop9 is an essential factor in the processing of preribosomal RNA. Its absence in yeast is lethal, and defects in the human ortholog are associated with breast cancer, autoimmunity, and learning/language impairment. PUF family RNA-binding proteins are best known for sequence-specific RNA recognition, and most contain eight α-helical repeats that bind to the RNA bases of single-stranded RNA. Nop9 is an unusual member of this family in that it contains eleven repeats and recognizes both RNA structure and sequence. Here we report a crystal structure of Saccharomyces cerevisiae Nop9 in complex with its target RNA within the 20S preribosomal RNA. This structure reveals that Nop9 brings together a carboxy-terminal module recognizing the 5' single-stranded region of the RNA and a bifunctional amino-terminal module recognizing the central double-stranded stem region. We further show that the 3' single-stranded region of the 20S target RNA adds sequence-independent binding energy to the RNA–Nop9 interaction. Both the amino- and carboxy-terminal modules retain the characteristic sequence-specific recognition of PUF proteins, but the amino-terminal module has also evolved a distinct interface, which allows Nop9 to recognize either single-stranded RNA sequences or RNAs with a combination of single-stranded and structured elements.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural basis of TFIIIC-dependent RNA polymerase III transcription initiation

RNA polymerase III (Pol III) is responsible for transcribing 5S ribosomal RNA (5S rRNA), tRNAs, and other short non-coding RNAs. Its recruitment to the 5S rRNA promoter requires transcription factors TFIIIA, TFIIIC, and TFIIIB. Here, we use cryoelectron microscopy (cryo-EM) to visualize the S. cerevisiae complex of TFIIIA and TFIIIC bound to the promoter. Gene-specific factor TFIIIA interacts with DNA and acts as an adaptor for TFIIIC-promoter interactions. We also visualize DNA binding of TFIIIB subunits, Brf1 and TBP (TATA-box binding protein), which results in the full-length 5S rRNA gene wrapping around the complex. Our smFRET study reveals that the DNA within the complex undergoes both sharp bending and partial dissociation on a slow timescale, consistent with the model predicted from our cryo-EM results. Our findings provide new insights into the transcription initiation complex assembly on the 5S rRNA promoter and allow us to directly compare Pol III and Pol II transcription adaptations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Birth of protein folds and functions in the virome

The rapid evolution of viruses generates proteins that are essential for infectivity and replication but with unknown functions, due to extreme sequence divergence. Here, using a database of 67,715 newly predicted protein structures from 4,463 eukaryotic viral species, we found that 62% of viral proteins are structurally distinct and lack homologues in the AlphaFold database. Among the remaining 38% of viral proteins, many have non-viral structural analogues that revealed surprising similarities between human pathogens and their eukaryotic hosts. Structural comparisons suggested putative functions for up to 25% of unannotated viral proteins, including those with roles in the evasion of innate immunity. In particular, RNA ligase T-like phosphodiesterases were found to resemble phage-encoded proteins that hydrolyse the host immune-activating cyclic dinucleotides 3',3'- and 2',3'-cyclic GMP-AMP (cGAMP). Experimental analysis showed that RNA ligase T homologues encoded by avian poxviruses similarly hydrolyse cGAMP, showing that RNA ligase T-mediated targeting of cGAMP is an evolutionarily conserved mechanism of immune evasion that is present in both bacteriophage and eukaryotic viruses. Together, the viral protein structural database and analyses presented here afford new opportunities to identify mechanisms of virus–host interactions that are common across the virome.

59 BASIC BIOLOGICAL SCIENCES↗