The FEBS Journal

Bacterial cells use multiple environmental cues to regulate levels of the second messenger cyclic dimeric GMP. This compound influences key lifestyle decisions such as motility, biofilm formation, or virulence. Although many diguanylate cyclases (DGCs) combined with various sensory domains have been studied previously, how distinct inputs are integrated within a single enzyme remains incompletely understood. Here, we investigate a cyanobacterial family of dual-sensor DGCs that combine an N-terminal receiver (Rec) domain followed by a light-oxygen-voltage (LOV) domain upstream of a diguanylate cyclase (GGDEF) domain. Using in vivo activity screening and in vitro characterisation, we determined how phosphorylation and blue light, individually and jointly, regulate enzyme activity. By measuring kinetic parameters across four defined functional states, unphosphorylated or phosphorylated, in combination with dark or light states, we reveal logic gate-like behaviours. One representative, LaRldC, integrates both signals with pronounced fold-changes in activity-, consistent with overall OR-type logic and with light acting as the dominant input. Our results demonstrate its function as a molecular gate coupling phosphorylation and illumination sensing to cyclic-di-GMP formation. These findings provide valuable insights into multi-signal decision-making in cyanobacteria and establish further understanding of how modular sensory domains are wired to control bacterial second-messenger signalling.

The RBR E3 ligase parkin is recruited to the outer mitochondrial membrane (OMM) during oxidative stress where it becomes activated and ubiquitinates numerous proteins. Parkin activation involves binding of a phosphorylated ubiquitin (pUb), followed by phosphorylation of parkin itself, both mediated by the OMM kinase, PINK1. However, targeted mitochondrial proteins have little structural or sequence similarity, with the commonality between substrates being proximity to the OMM. Here, we demonstrate that parkin efficiently ubiquitinates a mitochondrial acceptor pre-ligated to pUb and phosphorylation of parkin triggers autoubiquitination activity. Mitochondrial target proteins, Miro1 or CISD1, tethered to pUb are ubiquitinated by parkin more efficiently than if alone or Ub-tethered and ubiquitin molecules are ligated to acceptor protein lysines and not pUb. Parkin phosphorylation is not required for acceptor-pUb ubiquitination. In fact, only phospho-parkin induced self-ubiquitination and deletion of Ubl or mutation at K211N inhibited self-ubiquitination. We propose divergent parkin mechanisms whereby parkin-mediated ubiquitination of acceptor proteins is driven by binding to pre-existing pUb and subsequent parkin phosphorylation triggers autoubiquitination. This finding is critical for understanding parkins role in mitochondrial homeostasis and has implications on targets for therapeutics.

Wnt signalling is a cornerstone of embryonic development, orchestrating critical processes such as body axis formation, gastrulation, and organogenesis through conserved canonical and non-canonical pathways. Dishevelled (Dvl), a central mediator of these pathways, contains conserved DIX, PDZ, and DEP domains, along with an extreme-C-terminus. Recent studies suggest that the extreme-C-terminus regulates non-canonical Wnt signalling via an autoinhibitory interaction with the PDZ domain. Non-canonical Wnt signalling branches into the planar cell polarity (PCP) and Wnt/Ca{superscript 2} pathways. Profilin, a monomeric actin-binding protein, has been implicated in PCP signalling through Daam1-mediated actin polymerization, whereas its silencing disrupts the Wnt/Ca{superscript 2} pathway in a Daam1-independent manner, pointing to a role for profilin upstream of Daam1. In this study, we identify a novel interaction between Dvl2 and profilin2. Co-localization and in vitro pull-down assays demonstrate that profilin2 directly interacts with Dvl2. Furthermore, our study reveals profilin2 binds specifically to the extreme-C-terminus of Dvl2, beyond the polyproline motif, without engaging the PDZ or DEP domains. This challenges the conventional view of profilin-polyproline interactions and highlights the existence of previously unrecognized molecular determinants. Moreover, we show that Dvl2 adopts an autoinhibited conformation through intramolecular binding of its extreme-C-terminus to the PDZ domain. Remarkably, profilin2 retains its binding ability even in this autoinhibited state. Together, these findings uncover a previously unrecognized profilin2-Dvl2 interaction and provide new mechanistic insights into the molecular regulation of non-canonical Wnt signalling.

The expression of virulence factors essential for the invasion of host cells by Salmonella enterica is tightly controlled by a network of transcription regulators. The AraC/XylS transcription factor HilD is the main integration point of environmental signals into this regulatory network, with many factors affecting HilD activity. Long chain fatty acids (LCFAs), which are highly abundant throughout the host intestine directly bind to, and repress HilD, acting as environmental cues to coordinate virulence gene expression. The regulatory protein HilE also negatively regulates HilD activity, through a protein-protein interaction. Both of these regulators inhibit HilD dimerisation, preventing HilD from binding to target DNA. We investigated the structural basis of these mechanisms of HilD repression. LCFAs bind to a conserved pocket in HilD, in a comparable manner to that reported for other AraC/XylS regulators, whereas HilE forms a stable heterodimer with HilD by binding to the HilD dimerisation interface. Our results highlight two distinct mechanisms by which HilD activity is repressed, which could be exploited for the development of new antivirulence leads.

Type II NADH dehydrogenases (NDH-2s) are accessory enzymes of the bacterial electron transport chain (ETC). While they functionally overlap with Complex I, their main role is not proton translocation but maintaining the intracellular NADH/NAD+ balance. Although often non-essential, NDH-2 become crucial in species lacking complex I, serving as the primary electron entry point into the ETC. Their virtual absence in mammals makes these enzymes attractive targets for antimicrobial drug development and mitochondrial functional restoration. NDH-2s catalyze electron transfer from NADH to quinones, yet two distinct catalytic mechanisms have been described for members of the family: a classical ping-pong mechanism and an atypical ternary mechanism involving the formation of a charge transfer complex (CTC). The molecular basis of these mechanisms remains unclear. Also, their occurrence among NDH-2s from different bacterial lineages in unknown. Here we combined molecular phylogenetics, ancestral sequence reconstruction, expression and biochemical characterization of ancestral and modern enzymes and, molecular dynamics simulations to explore the mechanistic versatility of NDH-2s across Bacteria. Our results show the atypical ternary mechanism is restricted to the Firmicutes (Bacillota) lineage and it is defined by the presence of a single substitution located at the bottom of the active site. This work provides an evolutionary framework for understanding NDH-2 mechanistic versatility. Besides, it establishes a basis for drug discovery targeting pathogenic strains and opens avenues to develop innovative strategies to complement dysfunctional mitochondria.

Acetyl and other acyl groups from different short-chain fatty acids (SCFA) competitively modify histones at various lysine sites. To fully understand the functional significance of such histone acylation, a key epigenetic mechanism, it is crucial to characterize the cellular sources of the corresponding acyl-CoA molecules required for the lysine modification. Like acetate, SCFAs such as propionate, butyrate and crotonate are thought to be the substrates used to generate the corresponding acyl-CoAs by enzymes known as acyl-CoA synthetases. The acetyl-CoA synthetase, ACSS2, which produces acetyl-CoA from acetate in the nucleocytoplasmic compartment, has been proposed to also mediate the synthesis of acyl-CoAs such as butyryl- and crotonyl-CoA from the corresponding SCFAs. This idea is now widely accepted and is sparking new research projects. However, based on our direct in vitro experiments with purified or recombinant enzymes and structural considerations, we demonstrate that ACSS2 is unable to mediate the generation of non-acetyl acyl-CoAs like butyryl- and crotonyl-CoA. It is therefore essential to re-examine published data and corresponding discussions in the light of this new finding.

The proteolytic active sites of the 26S proteasome are sequestered within the catalytic chamber of its 20S core particle (CP). Access to this chamber is through a narrow channel defined by the outer subunits. Free 20S CP is found in a latent state in which the N-termini of neighboring subunits form a gate blocking access to the channel. Entry of substrates can be facilitated by the attachment of the activators or regulatory particles, which can rearrange the blocking subunit N-terminal residues. In order to determine the specific physiological role of individual elements working in concert within the gate, we constructed a set of truncations or single-site mutations in each of the participating N-terminal tails. We report herein that whereas only a few N-termini are important for maintaining a closed gate, all seven N-termini participate in the open gate. Specifically, an invariant tyrosine (Y) in each subunit forms a hydrogen bond with a conserved aspartate (D) in the N-terminal tail of its counterclockwise neighbor, with the exception of the 1-2 pair leaving a gap in the ring circumference. A third residue (X) of this YD(X) motif aligns the open channel; specifically, the phenylalanine (F) at this position of the 2 subunit comes in direct contact with the translocating substrate. Consequently, deletion of this 2 N-terminal tail slows down proteolysis despite the appearance of an open gate state. We conclude that the YD(X) motif in N-terminal tail of subunits plays an important role in gating the proteasome and in processing the substrate.

Despite several discoveries in recent years, the physiology of acidophilic Micrarchaeota remains largely enigmatic. "Candidatus Micrarchaeum harzensis A_DKE", for example, highly expresses numerous genes encoding hypothetical proteins and their function is difficult to elucidate due to a lacking genetic system. Still, not even the intracellular pH value of A_DKE is known, and heterologous production attempts are generally missing so far. Hence, A_DKEs isocitrate dehydrogenase (MhIDH) was recombinantly produced in Escherichia coli and purified for bio-chemical characterisation. MhIDH appeared to be specific for NADP+, yet promiscuous regarding divalent cations as cofactors. Kinetic studies showed KM-values of 53.03{+/-}5.63 {micro}M and 1.94{+/-}0.12 mM and kcat-values of 38.48{+/-}1.62 s-1 and 43.99{+/-}1.46 s-1 for DL-isocitrate and NADP+, respectively. MhIDHs exceptionally low affinity for NADP+, potentially limiting its reaction rate, can be likely attributed to the presence of a proline residue in the NADP+ binding-pocket, which might cause a decrease in hydrogen bonding of the cofactor and a distortion of local secondary structure. Furthermore, a pH optimum of 7.89 implies, that A_DKE applies potent mechanisms of proton homoeostasis, to maintain a slightly alkaline cytosolic milieu in a highly acidic environment.

The process of bacterial coenzyme A (CoA) degradation has remained unknown despite the otherwise detailed characterization of the CoA synthesis pathway over 30 years ago. Numerous enzymes capable of CoA degradation have been identified in other domains of life that belong to the Nudix superfamily of hydrolases, but the molecule responsible for this process in the model bacterial system of E. coli remains a mystery. We report here that E. coli contains two such Nudix enzymes capable of CoA degradation into 4-phosphopantetheine and 3,5-adenosine monophosphate. The E. coli enzymes NudC and NudL were cloned in various promoter-fusion constructs in order to purify them as soluble active enzymes and characterize their ability to catalyze the phosphohydrolysis of CoA. NudC, an enzyme known to hydrolyze NADH as its principal substrate, demonstrated the ability to hydrolyze CoA, among other coenzymes, at comparable rates to eukaryotic Nudix hydrolases. NudL, a previously uncharacterized enzyme, demonstrated the ability to cleave only CoA and CoA-related molecules at a rate orders of magnitude slower than its eukaryotic orthologs. NudC and NudL therefore represent a previously uncharacterized pathway of CoA degradation in the highly studied E. coli system. While the two enzymes display some substrate overlap, their respective activities imply that NudC may play a role as a general coenzyme hydrolase, while NudL specifically targets CoA. These data further suggest a role for these enzymes in the regulation of bacterial CoA-RNA.

AAA proteases are hexameric ATP-dependent metallopeptidases that perform crucial proteolytic activities within prokaryotic and eukaryotic membranes. Structurally, protomers are comprised of catalytically active C-terminal domains that are anchored to the membrane by an N-terminal autonomous folding unit. In this study, we determined the fold, stability, and oligomeric state of the N-terminal intermembrane domains of human spastic paraplegia type 7 (SPG7)/ paraplegin protein and its bacterial orthologue FtsH using circular dichroism (CD), small-angle X-ray scattering (SAXS), small-angle neutron scattering (SANS) and X-ray crystallography. Solution-state analysis revealed that the N-terminal domain of paraplegin is a monomer in solution whereas FtsH forms a dimer. Unexpectedly, the N-terminal domain of paraplegin presents as a domain-swapped homodimer in our crystal structure that involves the first helix and first two beta-strands from one monomer and beta-strand 3, helix 2 and beta-strand 4 from another symmetry-related molecule. However, together they form an assembly which is similar to protomers observed for the N-terminal regions of FtsH and AfG3L2. Drawing from our structural data, we postulate that domain-swapping interactions of the N-terminal regions contribute to stability of the AAA protease hexamer containing paraplegin, demonstrating the extensive flexibility of the N-terminal portion of this protein and its role in achieving the appropriate molecular architecture required for function. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/720153v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1f4b9b5org.highwire.dtl.DTLVardef@1cc2242org.highwire.dtl.DTLVardef@dd211borg.highwire.dtl.DTLVardef@1a87722_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIFtsH-IMS forms a homo-dimer in solution, whereas paraplegin-IMS presents as a well-folded monomer in solution C_LIO_LIparaplegin-IMS crystallises as a domain-swapped homo-dimer but its domain-swapped monomers are structurally similar to other IMS-regions C_LIO_LIAfG3L2/paraplegin hexamer formation may be supported by domain swapping in paraplegin-IMS C_LIO_LIdomain-swapping in paraplegin could be a Bonafide feature under certain cellular conditions and may be related to disease in spastic paraplegia C_LI

Only a few proteins (13 in humans) are encoded by the mammalian mitochondrial genome. Therefore, the other mitochondrial resident proteins (>1000) must be recruited via specialised import pathways. Protein import is critical for mitochondrial biogenesis and bioenergetic function and health; loss of function has been implicated with a wide range of pathologies. Despite this, our understanding of the kinetic and dynamics of import is somewhat limited, particularly within mammalian cells. Here, we report an adaptation of an assay system, established previously to monitor mitochondrial import into isolated yeast mitochondria, to quantitatively monitor mitochondrial import inside mammalian cells. The reporting is based on a split luciferase, whereby the large fragment is segregated in the mitochondrial matrix and the small complementary fragment is fused to the C-terminus of a recombinant precursor protein destined for import. Following import successively through the TOM complex of the outer membrane and the TIM23 complex of the inner membrane, the complementary fragments combine to form an active luciferase. The resultant luminescent signal provides a sensitive, accurate, free of noise and continuous measure of protein import, enabling mathematical model fitting to identify and understand the steps that make up import. This advance allows detailed mechanistic examination of the transport process in live cells. In addition, the assay will enable characterisation of the protein import when the machinery is challenged; for example, in situations associated with disease. Moreover, the assay is compatible with high throughput for large data set collection and kinetic modelling, as well as for drug screening and characterisation. Our set-up also has the potential to be adapted for the analysis of alternative transport systems and different cell types, and even for multicellular model organisms.

StatementThe authors have withdrawn our manuscript whilst we perform additional experiments to test some of our conclusions further. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

Ubiquitination is a post-translational modification responsible for one of the most complex multi-layered communication and regulation system in the cell. Over the past decades, new ubiquitin variants and ubiquitin-like proteins arose to further enrich this mechanism. Among them, the recently discovered ubiquitin variant UbKEKS can specifically target several proteins and yet, functional consequences of this new modification remain unknown. The absence of UbKEKS induces accumulation of lamin A in the nucleoli, highlighting the need for deeper investigations about protein composition and functions regulation of this highly dynamic and membrane-less compartment. By using data independent acquisition mass spectrometry and microscopy, we show here that despite not impacting protein stability, UbKEKS is required to maintain normal nucleolar organization. The absence of UbKEKS increases nucleolis size and accentuate their circularity while disrupting dense fibrillar component and fibrillar center structures. Moreover, depletion of UbKEKS leads to distinct changes in nucleolar composition. Notably, lack of UbKEKS favors nucleolar sequestration of known apoptotic regulators such as IFI16 or p14ARF, resulting in an increase of apoptosis in UbKEKS knockout cells observed by flow cytometry and real-time cellular growth monitoring. Overall, the results presented here identifies the first cellular functions of the UbKEKS variant and lay the foundation stone to establish UbKEKS as a new universal layer of regulation in the already complex ubiquitination system.

Varicella Zoster Virus (VZV) is a dsDNA virus that infects dermal cells and causes characteristic cutaneous lesions. The virus undergoes neurotropism and later causes secondary cycles of infection. In the host nucleus, Promyelocytic Leukaemia Nuclear Bodies (PML-NBs) spontaneously form around the VZV genome to repress viral gene expression. VZV encodes for a ubiquitin E3 ligase ORF61 to disperse PML-NBs and alleviate repression. ORF61 functions as a ubiquitin E3 ligase with a conserved RING domain at the N-terminal end. It carries three SUMO-interacting motifs (SIMs) that mediate interactions with SUMOylated proteins within PML bodies. The mechanism by which ORF61 disperses PML-NBs is poorly understood. To understand how ORF61 interacts with SUMOylated proteins, we investigated its interaction with SUMO and studied its SUMO-Targeted Ubiquitin Ligase (STUbL) activity. Our studies reveal that ORF61 co-opts the E2D family for ubiquitination activity. A specific network of interactions between the E2 enzyme, ORF61, and Ub facilitates polyubiquitination. ORF61 can synthesize branched polyubiquitin chains of K11, K48, and K63 linkages. The C-terminal SIM in ORF61 is a high-affinity binder of SUMO chains. Utilizing the SIM, ORF61 targets specific lysines on SUMO chains for ubiquitination. These studies provide crucial insights into the functional mechanism of viral STUbL ORF61.

Trimethylation of lysine 4 of histone H3 (H3K4me3) is a post-translational modification (PTM) enriched at promoters of actively transcribed genes. H3K4me3 is removed by the human histone demethylases of the KDM5 family. KDM5 demethylases act as transcriptional repressors through their catalytic activity in addition to more complex roles that depend on their interactions with other chromatin regulators and may be independent of demethylase activity. To better understand the mechanistic differences of the closely related paralogs KDM5A and KDM5B as well as their interactions with Retinoblastoma protein (RB), we systematically analyzed and compared their demethylase activities, nucleosome engagement, and RB binding. We used recombinant nucleosome binding and demethylase activity assays, as well as an integrative structural biology approach using negative-stain electron microscopy (EM), AlphaFold predictions, and cross-linking mass spectrometry for a comprehensive in vitro analysis of these critical and largely non-redundant enzymes. KDM5A and KDM5B showed differences in enzyme kinetics using peptide substrates, as well as in nucleosome binding. Furthermore, KDM5A interacts with RB, mainly mediated by its canonical LxCxE RB binding motif. KDM5B, on the other hand, lacks an LxCxE binding motif and does not stably bind to RB under the conditions tested here. RB directly interacts with nucleosomes, and its nucleosome binding does not measurably affect KDM5A demethylase activity or nucleosome interactions. Our findings provide a biochemical framework for the differences between KDM5A and KDM5B regarding RB interactions and nucleosome engagement.

Parasitic nematodes infect billions of humans and livestock worldwide, causing major health and economic burdens, while the spread of anthelmintic resistance threatens current control strategies. A critical step in parasite infection is the resumption of development of infective third-stage larvae (iL3) upon host entry, a process controlled by the nuclear receptor DAF-12. Activation of DAF-12 by dafachronic acids promotes developmental progression and reproductive maturation, making this receptor an attractive therapeutic target. However, the molecular mechanisms governing DAF-12 activation, particularly transcriptional coactivator recruitment, remain poorly understood. Here, we combined biophysical, cellular, structural, and bioinformatic approaches to investigate coactivator recognition by DAF-12 from the parasitic nematodes Brugia malayi and Haemonchus contortus. Crystal structures of ligand-bound DAF-12 ligand-binding domains in complex with coactivator-derived peptides reveal conserved features of ligand-dependent coactivator recruitment shared with mammalian nuclear receptors. In addition, we uncover previously unrecognized interaction features, including motif-specific contacts that extend beyond the canonical LXXLL binding mode of coactivators and distinct patterns of DAF-12 conservation across nematode clades. Structure-guided analyses redefine the interaction motif of the only described parasite-specific coactivator DIP-1 and suggest novel candidate motifs for DAF-12-interacting proteins. Together, these findings establish the structural basis of coactivator binding to nematode DAF-12 and provide mechanistic insight into the transcriptional regulation underlying parasite development. These results expand current understanding of nuclear receptor signaling in parasitic nematodes and provide a framework for the future design of strategies aimed at disrupting DAF-12 activation as a potential antiparasitic approach. Author SummaryParasitic nematodes infect billions of people and livestock worldwide, causing major health and economic burdens, while increasing resistance threatens current treatments. These parasites rely on a developmental switch that allows infectious larvae to resume growth inside their host, a process controlled by the nuclear receptor DAF-12. Blocking this pathway could prevent parasites from establishing infection. However, the molecular mechanisms regulating DAF-12 activation remain poorly understood. Here, we investigate how DAF-12 from two parasitic nematodes, Brugia malayi and Haemonchus contortus, interacts with transcriptional coactivators that enable gene activation, using a combination of biophysical, cellular, structural, and bioinformatic approaches. We identified conserved features of ligand-dependent coactivator recruitment shared with mammalian nuclear receptors as well as nematode-specific interaction mechanisms that vary across evolutionary clades. These findings provide new insights into the structural basis of coactivator binding to DAF-12 and advance our understanding of a key pathway controlling parasitic nematode development.

Adenylyl cyclases (ACs) and their catalytic product cAMP are regulatory components of plant responses. AC domains are intrinsic components of complex molecules with multiple functions, some of which are co-regulated by cAMP. Here we used an amino acid search motif based on annotated ACs in organisms across species to identify 12 unique Arabidopsis thaliana candidate ACs, four of which have a role in the biosynthesis of the stress hormone abscisic acid (ABA). One of these, the 9-cis-epoxycarotenoid dioxygenase (NCED3, At3g14440), was identified by sequence and structural analysis as a putative AC and then tested experimentally for activity. We show that an NCED3 AC fragment can complement an AC deficient E. coli mutant and this rescue is nullified when key amino acids in the AC motif are mutated. AC activity was also confirmed by tandem liquid chromatography mass spectrometry (LC-MS/MS). Our results are consistent with a moonlighting role for mononucleotide cyclases in multi-domain proteins that have at least one other distinct molecular function such as catalysis or ion channel activation and promise to yield new insights into tuning mechanisms of ABA dependent plant responses. Finally, our search method can also be applied to discover ACs in other species including Homo sapiens. HighlightsO_LIAn adenylyl cyclase (AC) catalytic center motif identifies novel ACs in plants C_LIO_LIACs can moonlight in complex proteins with other enzymatic domains C_LIO_LIA 9-cis-epoxycarotenoid dioxygenase essential for abscisic acid synthesis contains an AC C_LIO_LIThis finding implicates cAMP in abscisic acid synthesis and signaling C_LI

The ubiquitin-like modifier ISG15 can modulate host and viral proteins to restrict viral and microbial infections, and act as a cytokine. Its expression and conjugation are strongly up-regulated by type I interferons. Here we identify the deubiquitinating enzyme USP16 as an ISG15 cross-reactive protease. Ubiquitin-specific protease 16 (USP16) was found to react with an ISG15 activity-based probe in pull-down experiments using chronic myeloid leukaemia-derived human cells (HAP1). Supporting this finding, recombinant USP16 cleaved pro-ISG15 and ISG15 iso-peptide linked model substrates in vitro, as well as ISGylated substrates present in cell lysates. Moreover, the interferon-induced stimulation of ISGylation in human HAP1 cells was increased by knockdown or knockout of USP16. Depletion of USP16 did not affect interferon signaling, and interferon treatment did not affect USP16 expression or enzymatic activity either. A USP16-dependent ISG15 interactome was established by anti-ISG15 immunoprecipitation mass spectrometry (IP-MS), which indicated that the deISGylating function of USP16 may regulate metabolic pathways involving GOT1, ALDOA, SOD1 and MDH1, all of which were further confirmed to be deISGylated by USP16 in HEK293T cells. Together, our results indicate that USP16 may contribute to regulating the ISGylation status of a subset of proteins related to metabolism during type I interferon responses.

Toll-like and Interleukin-1/18 receptor resistance (TIR) domain-containing proteins function as important signaling and immune regulatory molecules. TIR domain-containing proteins identified in eukaryotic and prokaryotic species also exhibit NAD+ hydrolase activity in select bacteria, plants, and mammalian cells. We report the crystal structure of the Acinetobacter baumannii TIR domain protein (AbTir-TIR) with confirmed NAD+ hydrolysis and map the conformational effects of its interaction with NAD+ using HDX-MS. NAD+ results in mild decreases in deuterium uptake at the dimeric interface. In addition, AbTir-TIR exhibits EX1 kinetics indicative of large cooperative conformational changes which are slowed down upon substrate binding. Additionally, we have developed label-free imaging using 2pFLIM which shows differences in bacteria expressing native and mutant NAD+ hydrolase-inactivated AbTir-TIREA protein. Our observations are consistent with substrate-induced conformational changes reported in other TIR model systems with NAD+ hydrolase activity. These studies provide further insight into bacterial TIR protein mechanisms and their varying roles in biology.

Withdrawal noticeThe authors have withdrawn their manuscript. The lipidomic data presented in the manuscript were based on an excel summary sheet provided by VIT, whose group (AP and KM) conducted the analysis using an Orbitrap mass spectrometer. However, after submitting to BioRxiv, the VIT group could not recover the RAW (primary source) files from the lipidomic platform because these files were deleted due to the maintenance protocol used for the Orbitrap mass spectrometer. It was agreed to reconduct the lipidomic analysis. As the Orbitrap mass spectrometer was out of service at that time, the lipidomic analysis was conducted with the help of a QToF mass spectrometer. Important differences were noted for the relative abundance and species of many lipids across the strains compared to the previously reported data hence casting some doubt on their interpretation. Therefore, until further analysis can be carried out the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.