Metabolic homeostasis depends on adaptive control of intracellular metabolite flux, yet how such control is reconfigured when canonical transport pathways fail is unknown. Here we define a conserved vesicular circuit that preserves systemic heme balance by rerouting intracellular heme flux. We show that loss of the intestinal heme exporter MRP-5 in Caenorhabditis elegans causes lethal heme sequestration within endolysosomal compartments. This defect is bypassed by disabling the vesicular adaptor AP-3, which stabilizes and reroutes the heme importer HRG-1, restoring heme export without increasing cytosolic heme. Unbiased genetics and transcriptomics identify two previously uncharacterized SLC49A family members, HRG-13 and HRG-14, as heme exporters with distinct affinities that engage in a vesicular importer-exporter handoff. Live imaging reveals heme-enhanced contacts between HRG-1 and SLC49A-containing vesicles, consistent with direct vesicular transfer. This circuitry extends to vertebrates as disruption of the SLC49A3 homolog impairs erythropoiesis in zebrafish and causes intracellular heme overload, premature hemoglobinization and apoptosis in differentiating human erythroid cells. Together, these findings establish SLC49A3 proteins as conserved heme exporters and uncover a general principle of metabolic adaptation in which reprogramming intracellular compartmentalization, rather than increasing nutrient supply, restores systemic homeostasis.

Biological processes rely on finely tuned homo- and heteromeric interactions between (biomacro)molecules. The strength of an interaction, typically given by the dissociation constant (K D), plays a crucial role in basic research and must be monitored throughout the development of drugs and agrochemicals. An ideal method for K D determination is applicable to various analytes with a large range of affinities, tolerates complex matrix compositions, does not require labeling, and simultaneously provides information on the structural integrity of the binding partners. Native mass spectrometry meets these criteria but typically struggles with homooligomeric complexes due to overlapping mass signals. To overcome this, we resolve monomer/dimer contributions to overlapping MS-peaks by separately analyzing the charge state distribution of each oligomeric species via sample dilution and covalent cross-linking. Following this approach, we show that quantitative laser-induced liquid bead ion desorption mass spectrometry (qLILBID-MS) accurately captures the affinities of Bovine Serum Albumin (BSA) and chemically induced dimers of Tryparedoxin (Tpx), an oxidoreductase from human pathogenic Trypanosoma brucei parasites, with various molecular glues and homodimer affinities. Conveniently, qLILBID-MS requires a fraction of sample used by other methods such as isothermal titration calorimetry (ITC) and yields previously inaccessible protein homodimer K Ds in the high micromolar range, which allowed us to monitor the gradual decrease in homodimer affinity via mutation of crucial dimer interface contacts. Overall, qLILBID-MS is a sensitive, robust, fast, scalable, and cost-effective alternative to quantify protein/protein interactions, that can accelerate contemporary drug discovery workflows, e.g. the efficient screening for proximity inducing molecules like proteolysis targeting chimera (PROTACs) and molecular glues.

Peptidases are indispensable tools in biotechnology and chemical biology. However, the enzyme repertoire for the selective hydrolysis of d l-amide bonds in peptides is small. Here, we describe novel dl-peptidases that mediate complex microbial interactions. These enzymes, Lip3 and Lip7, convert lipopeptides into potent amoebicidal agents via selective d l-peptide bond cleavage. Using structural analyses and mutagenesis, we identified an unusual Ser-Lys-Lys-Tyr catalytic tetrad required for dl-specificity. Despite their high structural similarity, both enzymes show distinct substrate preferences: Lip3 acts primarily as a carboxypeptidase, removing a single C-terminal residue, while Lip7 excises a tripeptide. Although their substrate scopes are broad, they are highly specific with regard to their respective cutting sites. These features make these dl-peptidases powerful tools for elucidating the structure of complex peptide-based natural products, including tensin and WLIP. Overall, this work elucidates the molecular mechanisms of cooperative microbial defense and provides a new enzymatic toolbox for biocatalysis and natural product discovery.

Bacteriophages (phages) play essential roles in microbial systems, yet most phage proteins remain poorly characterised. Protein tertiary and quaternary structure information contributes valuable information about protein function. As many phage proteins function as homooligomers, complexes that consist of multiple identical subunits, there is great interest in computationally predicting their configurations. Here we present a computational framework, the Phage Homomer Level Estimate and Generation Method (PHLEGM) for inferring homooligomeric states directly from the protein sequence by combining AlphaFold-Multimer modelling with inter-subunit interface quality assessment. We proceeded to experimentally validate two out of nine predicted homooligomers using size exclusion chromatography and complementary hydrodynamic techniques. These efforts confirmed our predictions for a dimer and a trimer, highlighting the value of experimentally benchmarked computational predictions and showing the challenges of heterologous phage protein production. Applied to >22,000 phage protein sequences in the PHROGs database, our approach revealed extensive diversity in phage homooligomeric protein complexes. Benchmarking against protein language model-based predictors on a curated reference set of known phage homooligomers demonstrated superior accuracy of our structure-based method, achieving robust performance in classifying protein homooligomeric states, with the highest accuracy observed for trimers and higher-order complexes. These results highlight the value of computational predictions to decipher the complexities of the vast viral sequence space. All predicted complex structures and functional inferences are made publicly available to support structural and functional studies of phage proteins.

Ti-6Al-4V, a widely used titanium alloy for load-bearing implants, is rapidly conditioned by adsorbed proteins in vivo. Protein conditioning layers on Ti-6Al-4V implants strongly influence subsequent cell and microbial interactions, yet reproducible process parameters for well-defined human serum albumin (HSA) coatings are still lacking. Ethanol-water mixtures drive conformational transitions of HSA, but it remains unclear when β-rich, thioflavin‑T (ThT)-positive HSA solution states translate into laterally connected protein nanofiber (PNF) coatings on Ti‑6Al‑4V. Here, we map ethanol-driven HSA structural transitions and PNF formation across ethanol fraction (20-80% v/v), HSA concentration (500-1500 µg·mL-1) and incubation time (6-72 h), and refine the effects of temperature (65-85 °C) and pH (3/7.4/10). Solution-state readouts comprise time-resolved ThT fluorescence (ThT kinetics) and far-UV circular dichroism (CD) spectroscopy measured on a selected subset of conditions. They are linked to surface outcomes using whole‑disc ThT-positive area fraction, atomic force microscopy (AFM)‑resolved fibrillar coverage/connectivity, and X-ray photoelectron spectroscopy (XPS). We found that β‑rich solution-state signatures of HSA form readily under acidic conditions, yet β‑sheet enrichment is not sufficient for a connected coating: at 60% ethanol and 65 °C, pH 10 produces a percolating fibrillar mesh with high AFM fiber coverage, whereas pH 3 yields sparse fibers despite substantial ThT‑positive surface area. The endpoint morphology and chemistry data support an operational surface-assisted assembly model in which adsorption/retention and lateral reorganization at the Ti-6Al-4V interface govern whether β-active deposits develop into a connected fibrillar network. The resulting parameter-process map provides design parameters for reproducible HSA-derived PNF coatings on Ti-6Al-4V and a framework for systematic studies of protein-conditioned implant interfaces.

Amoebal predation exerts a strong evolutionary selection pressure on bacteria, thus driving the development of effective predator-defense strategies. However, little is known about the molecular interplay between bacteria and predators, particularly how bacteria can sense and kill their microbial predators. We show how the ubiquitous bacterium Pseudomonas syringae detects and kills the social amoeba Polysphondylium pallidum. Combining comparative genomics, molecular biology, and chemical analyses, we identified a chemical radar system. The system relies on P. syringae secreting the lipopeptide syringafactin, which is deacylated by the amoeba. The resulting peptides are sensed via the bacterial sensor protein chemical radar regulator (CraR) that activates genes for converting the predator-derived signal into the amoebicide pyrofactin. This system is widespread in P. syringae and enables bacteria to infect A. thaliana in the presence of amoebae. Our study advances the understanding of microbial sensing and opens new avenues for the discovery of natural products.

Over 7 million people worldwide are affected with Chagas disease, a lifelong debilitating and potentially fatal Neglected Tropical Disease caused by the single cell protozoan parasite Trypanosoma cruzi. To maintain viability and to reproduce under the harsh conditions within a host organism, pathogens express a variety of protecting enzymes and virulence factors that can serve as potential drug targets. To protect itself from redox stress, T. cruzi takes advantage of a unique thiol metabolism. For instance, a cytosolic peroxide clearance cascade is centered on the conserved oxidoreductase Tryparedoxin (Tpx). Tpx efficiently distributes reducing equivalents across the parasitic cell through the promiscuous yet selective binding of numerous up- and downstream clients. However, the exact structure and binding interfaces of this central protein binding hub remain unknown. To study the redox-dependent structural dynamics of T. cruzi Tpx, and its interactions with binding partners, we determined the 1H, 13C, 15N-backbone NMR assignments of the enzyme in the reduced and oxidized state. In agreement with earlier NMR studies on Tpx from related protozoans, we report redox-dependent changes in the enzyme’s dithiol active site that could play a crucial role in the recognition of physiological substrates and should be considered in the rational design of small molecule inhibitors.

Macrophage infectivity potentiator (MIP) proteins, found in pro- and eukaryotic pathogens, influence microbial virulence, host cell infection, pathogen replication, and dissemination. MIPs share an FKBP (FK506 binding protein)-like prolyl-cis/trans-isomerase domain, making them attractive targets for inhibitor development. We determined high-resolution crystal structures of Burkholderia pseudomallei and Trypanosoma cruzi MIPs in complex with fluorinated pipecolic acid inhibitors. The inhibitor binding profiles in solution were compared across B. pseudomallei, T. cruzi, and Legionella pneumophila MIPs using 1H, 15N, and 19F NMR spectroscopy. Demonstrating the versatility of fluorinated ligands for characterizing inhibitor complexes, 19F NMR spectroscopy identified differences in ligand binding dynamics across MIPs. EPR spectroscopy and SAXS further revealed inhibitor-induced global structural changes in homodimeric L. pneumophila MIP. This study demonstrates the importance of integrating diverse methods to probe protein dynamics and provides a foundation for optimizing MIP-targeted inhibitors in this structurally conserved yet dynamically variable protein family.

Bacterially produced antimicrobial peptides (AMPs), or bacteriocins, play key roles in shaping microbial communities via interspecies competition. Unlike the more temporally dynamic gut microbiome, the oral microbiome exhibits long-term stability and is preserved into deep time in dental calculus, enabling evolutionary analysis across time. Here, we combine metagenomics, structural modeling, and experimental validation to investigate AMP diversity in ancient and modern dental biofilms from humans, Neanderthals, and nonhuman primates spanning 100,000 years. Using our newly developed platform, AMPcombi, we uncover evolutionary trajectories of bacteriocins and elucidate their ecological functions. Among these, we identify a conserved family of Actinomyces-derived defensin-like peptides, termed actifensins, present across all time periods. Phylogenetic, structural, and functional analyses revealed shared ancestry and adaptive diversification between ancient (paleo-) and modern actifensins, with evidence of positive selection and maintained antimicrobial activity. Our findings position the oral microbiome as a valuable reservoir for natural product discovery. In the face of rising antimicrobial resistance, evolutionary insights into AMP function open a door to next-generation therapeutics. AMPcombi streamlines this process, linking ancient biomolecules with biotechnology.

Phosphodiester groups occur ubiquitously in nature, e.g. in nucleic acids or in cyclic (di-)nucleotides important for signal transduction. Proteins often use polar or positively charged amino acids to interact with the negatively charged phosphodiester groups via hydrogen bonds and salt bridges. In contrast, the acidic amino acids aspartate and glutamate are generally not considered as important determinants for phosphodiester group recognition. Instead, they are regarded as detrimental to such interactions due to the assumed charge repulsion between their deprotonated, negatively charged side chain carboxylate groups and the phosphodiester. Accordingly, acidic amino acids are often purposefully introduced into proteins to abrogate nucleic acid interactions in functional studies. Here, we show that in appropriate structural contexts, glutamate side chains are readily protonated even at neutral pH and act as hydrogen bond donors to phosphodiester groups using a c-di-GMP binding protein - the GSPII-B domain of PilF from Thermus thermophilus - as an example. Surveying available RNA-protein and DNA-protein complex structures in the PDB, we found that hydrogen bonds between apparently protonated carboxylate groups of glutamate and aspartate and phosphodiester groups occur frequently in many different functional protein classes. Thus, the functional role of acidic amino acids in phosphodiester group recognition needs to be re-evaluated.

The 5'-3' exonuclease phospholipase D3 (PLD3) is a single-pass transmembrane protein undergoing sequential post-translational modifications (PTM) by N-glycosylation, AMPylation and proteolytic cleavage. The substrates of PLD3 5'-3' exonuclease activity are single-stranded DNAs and RNAs, which act as ligands for Toll-like receptors (TLRs) and trigger a downstream pro-inflammatory response. Although PLD3 has primarily been studied in immune cells, recent findings indicate its enrichment in neurons, where it plays a role in regulating axonal fitness in Alzheimer's disease (AD). However, the regulatory mechanisms governing the proteolytic processing of PLD3 into its catalytically active soluble form and its functional roles in both immune and neuronal cells remain unclear. Here, we describe the functional implications of PLD3 AMPylation, its direct interaction with the protein adenylyltransferase FICD, and changes in PLD3 processing in Parkinson's disease (PD) patient-derived neurons. We identified PLD3 AMPylation sites within the proteins' soluble region and show that mutation of these sites lead to loss of PLD3 exonuclease catalytic activity. FICD AMP-transferase accelerates PLD3 degradation and induces cellular stress response. Furthermore, depletion of the two human AMP-transferases FICD and SelO point towards a complex regulatory network governing PLD3 AMPylation. Together, our findings demonstrate a critical role of AMPylation in PLD3 processing and regulation of its catalytic activity and provide new insights into the protein's transport and localization to lysosomes. The observation that PLD3 regulation in PD-derived neurons is altered compared to healthy neurons further highlights its role in neurodegenerative diseases. ### Competing Interest Statement The authors have declared no competing interest.

The ankyrin repeat is one of the most abundant protein-protein interaction motifs in eukaryotes yet occurs in only a small number of ion channels. These channels are all members of the transient receptor potential (TRP) superfamily and contain prominent ankyrin repeat domains (ARDs) in their cytoplasmic N termini. In transient receptor potential vanilloid 4 (TRPV4), the importance of this domain has been highlighted by the finding that gain-of-function neuromuscular disease-causing missense mutations cluster on the ARD surface. Little is known currently about the extent of the TRPV4-ARD interactome, nor how it may be altered by disease-causing mutations. Here, we utilized a human proteome microarray to profile the ARD interactomes of WT and mutant TRPV4. Probing of the microarray with TRPV4WT-ARD revealed 78 interactors, including proteins related to ubiquitination and small GTPase signaling, such as the ubiquitin ligase NEDD4L and the RhoGEF ARHGEF10. In parallel experiments, we also identified the deubiquitinase OTUB2 as an interactor of the proximal N terminus. Comparison of the ARD interactomes of WT and mutant TRPV4 revealed 21 interactions affected by disease-causing mutations. Strikingly, one of these interactors, ARHGEF10, is also mutated in neuromuscular disease. Cell-based studies confirmed that ARHGEF10 exhibits a reduced capacity to coimmunoprecipitate with mutant TRPV4. Furthermore, calcium imaging studies demonstrated that ARHGEF10 overexpression suppressed TRPV4WT channel activity, but that this inhibition is abrogated by disease-causing mutations. Together, these findings provide insights into the functional roles of an ion channel ARD, as well as their disruption in disease, and offer a resource for future cell-based studies.

Chemically induced dimerization of proteins is a powerful approach to regulate biomolecular functions through small molecule ligands acting as "molecular glues". Here, we demonstrate that simple, thienopyrimidinone scaffold-based inhibitors efficiently promote homodimerization of an essential oxidoreductase from the human pathogenic parasite Trypanosoma brucei through selective covalent attachment and self-assembly. A fluorine walk strategy, commonly used to optimize small molecule properties, resulted in tuning induced dimer affinity across two orders of magnitude. NMR spectroscopy, MD simulations, chromatography, multi-angle light scattering, mass spectrometry, calorimetry, and functional assays reveal how the inhibitor fluorination pattern alters the dynamics and interactions of the enzyme-bound inhibitor and surface-exposed aromatic protein side chains, affecting both enzyme inhibition kinetics and induced dimerization. This work highlights how site-specific fluorination can modulate protein interactions and offers a framework for the design of novel molecular glues with broad applications in chemical biology and drug development.

ATP-binding cassette (ABC) transporters use ATP to transport substrates across membranes. In type IV ABC transporters, which include many multidrug resistance (MDR) pumps, communication between nucleotide-binding domains (NBDs) and transmembrane domains (TMDs) is mediated via large intracellular domains containing 'coupling helices'. However, how ATP hydrolysis and substrate transport are functionally coordinated remains unclear. In the bacterial type IV MDR transporter BmrA, we identify a conserved residue cluster at the NBD/TMD interface centered on W413. Mutation of this tryptophan uncouples ATP hydrolysis from transport activity. Mutagenesis, functional assays, nuclear magnetic resonance spectroscopy, hydrogen-deuterium exchange mass spectrometry, and photo-induced electron-transfer fluorescence correlation spectroscopy show that the cluster forms a bidirectional communication hinge that relays signals between the NBD and TMD via coupling helix 2. Hinge mutations affect both local and global dynamics thereby influencing transporter activity. These findings uncover an allosteric pathway critical for functional coupling in multidomain ABC transporters.

Biological processes rely on finely tuned homo- and heteromeric interactions between (biomacro)molecules. The strength of an interaction, typically given by the dissociation constant (KD), plays a crucial role in basic research and must be monitored throughout the development of drugs and agrochemicals. An ideal method for KD determination is applicable to various analytes with a large range of affinities, tolerates complex matrix compositions, does not require labeling, and simultaneously provides information on the structural integrity of the binding partners. Native mass spectrometry meets these criteria but typically struggles with homooligomeric complexes due to overlapping mass signals. To overcome this, we resolve monomer/dimer contributions to overlapping MS-peaks by separately analyzing the charge state distribution of each oligomeric species via sample dilution and covalent crosslinking. Following this approach, we show that quantitative Laser-Induced Liquid Bead Ion Desorption mass spectrometry (qLILBID-MS) accurately captures the affinities of Bovine Serum Albumin and chemically induced dimers of Tryparedoxin, an oxidoreductase from human pathogenic Trypanosoma brucei parasites, with various molecular glues and homodimer affinities. Conveniently, qLILBID-MS requires a fraction of sample used by other methods such as isothermal titration calorimetry and yields previously inaccessible protein homodimer KDs in the high micromolar range, which allowed us to monitor the gradual decrease in homodimer affinity via mutation of crucial dimer interface contacts. Overall, qLILBID-MS is a sensitive, robust, fast, scalable, and cost-effective alternative to quantify protein/protein interactions that can accelerate contemporary drug discovery workflows, e.g. the efficient screening for proximity inducing molecules like proteolysis targeting chimera and molecular glues.

Microbial polyketides represent a structurally diverse class of secondary metabolites with medicinally relevant properties. Aromatic polyketides are produced by type II polyketide synthase (PKS) systems, each minimally composed of a ketosynthase-chain length factor (KS-CLF) and a phosphopantetheinylated acyl carrier protein (holo-ACP). Although type II PKSs are found throughout the bacterial kingdom, and despite their importance to strategic bioengineering, type II PKSs have not been well-studied in vitro. In cases where the KS-CLF can be accessed via E. coli heterologous expression, often the cognate ACPs are not activatable by the broad specificity Bacillus subtilis surfactin-producing phosphopantetheinyl transferase (PPTase) Sfp and, conversely, in systems where the ACP can be activated by Sfp, the corresponding KS-CLF is typically not readily obtained. Here, we report the high-yield heterologous expression of both cyanobacterial Gloeocapsa sp. PCC 7428 minimal type II PKS (gloPKS) components in E. coli, which allowed us to study this minimal type II PKS in vitro. Initially, neither the cognate PPTase nor Sfp converted gloACP to its active holo state. However, by examining sequence differences between Sfp-compatible and -incompatible ACPs, we identified two conserved residues in gloACP that, when mutated, enabled high-yield phosphopantetheinylation of gloACP by Sfp. Using analogous mutations, other previously Sfp-incompatible type II PKS ACPs from different bacterial phyla were also rendered activatable by Sfp. This demonstrates the generalizability of our approach and breaks down a longstanding barrier to type II PKS studies and the exploration of complex biosynthetic pathways.