Vanadium-dependent haloperoxidases are enzymes found in bacteria, fungi and red or brown macroalgae. They are used as a defense system for the latter by producing bromoform. The intermediate product, HOBr or HOCl, is also a microbicidal compound that can be exploited in many domains as medical tools or disinfectant sprays for example. We show that the mutation identified in Ohshiro’s work, which alters substrate specificity toward chloride, is transferable to the homologous enzyme from Chondrus crispus. We then applied genetic and enzymatic engineering to design a chimera with glucose oxidase, thereby enhancing antimicrobial properties by providing a local source of H2O2. We used the SpyTag/SpyCatcher technology to form the chimera and obtained homogenous objects, indicating that one oligomeric form is favored. We demonstrated the release of HOBr and HOCl thanks to NADH that reacts spontaneously with it outside the active site of the enzyme. We also measured the steady-state kinetic parameters of the wild-type or modified enzymes. The formation of the chimera increased the specificity of ccVHPO1 inside the chimera, towards KBr or H2O2, compared to the enzyme alone with the SpyCatcher. Finally, we showed a significant increase in the microbicidal effect between a coupled enzymatic system with the glucose oxidase (the two enzymes are free in solution) compared to the chimera system, which is completely bactericidal at concentrations around 20 nM.

Multidomain proteins connected by flexible linkers populate conformational ensembles that are challenging to characterize using conventional structural biology methods. In domain–linker–domain (DLD) proteins, linker-mediated inter-domain relative positions and orientations are functionally relevant, yet their dynamical behavior in solution normally remain poorly described. Small-angle X-ray scattering (SAXS) provides ensemble-averaged structural information for such systems; however, coupling with computational modeling is required to accurately describe the dynamic behavior of this family of proteins in solution. Here, we present a systematic evaluation of five ensemble-generation strategies applied to a set of eighteen proteins sharing the same two globular domains, connected by naturally occurring linkers of varying length and composition. Modeling methods based on different underlying principles are compared by assessing their agreement to experimental SAXS data, showing a large disparity and systematic structural biases among them. Furthermore, for each approach, we examine the effect of refinement against SAXS restraints and assess its capacity to describe the experimental data, as well as the induced biases in global dimensions and inter-domain distance distributions. This analysis underlines the importance of the initial conformational pool for deriving experimentally compatible ensembles. Overall, this work provides a high-quality benchmark for SAXS-driven ensemble modeling of flexible, multidomain proteins and establishes a framework for the critical interpretation of solution scattering data in systems with pronounced conformational heterogeneity.

The spontaneous formation of a model protein corona was studied on nanoplastics, considered as model nanoparticles of the smallest fragments resulting from polyethylene (PE-NPs), which is the most abundant plastic environmental pollutant. Well-individualized monomeric BSA proteins and 30 nm radius PE-NPs, were mixed at different concentration ratios in a phosphate buffer and studied using Small-Angle X-ray Scattering (SAXS) experiments. The average number of proteins adsorbed per particle, their hydration, as well as concentration of free proteins remaining in solution is measured by fitting the whole SAXS data in absolute scale of the intensity [cm-1] over a large q-range. In this way, protein adsorption isotherms were built and show a good agreement with the Langmuir monolayer model. We found a higher affinity in the case of pristine PE-NPs (3.15 L.g-1) compared to PE-NPs stabilized with traces of pegylated surfactants (0.41 L.g-1), in line with previously reported values for nanoparticles of a different nature such as SiO2 or polystyrene (PS). The protein affinity depends on the PE particle size and surface state. At physiological pH, a dense BSA corona formed on pristine PE-NPs with a maximum amount of adsorbed proteins at the monolayer's saturation 1.5 mg.m-2, that compares well to pristine PE microplastic measurements. Such tailored and stable PE-NPs with a controlled protein corona are suitable for a wide range of applications, including the study of their fate in biotic and abiotic environments.

Many RNA-binding proteins (RBP) have been associated to several neurodegenerative diseases for which RBP-rich cytoplasmic inclusions represent a major histological hallmark. However, among RBPs, the occurrence with which TDP-43, a nuclear mRNA-binding protein, is detected in cytoplasmic inclusions is exceptionally high. To unravel the underlying mechanisms, we focus our analysis on the structured N-terminal domain (NTD) of TDP-43, which is distinct among RBPs as this domain mostly initiates TDP-43 homotypic interactions. Through an in depth structural analysis, we successively show that the cooperative binding of TDP-43 along long GU-rich intronic sequences antagonizes NTD/NTD interactions between adjacent TDP-43 along mRNA. In contrast, the TDP-43 cooperativity facilitates NTD/NTD interactions between TDP-43 located on distinct GU-rich sequences. We hypothesize that NTD/NTD interactions between distinct GU-rich sequences efficiently allow the compaction of long introns in neurons under physiological conditions. However, when the binding of TDP-43 to RNA is discontinuous because of a lack of cooperativity, aberrant NTD/NTD interactions between adjacent TDP-43 take place, promoting the aggregation of TDP-43 RRMs (RNA Recognition Motifs) under stress conditions. Altogether, we provide a detailed view of the physiological assembly of TDP-43 on introns and the putative weaknesses of TDP-43 that makes it distinct in its propensity for aggregation compared to other RBPs.

Monoclonal antibodies recognizing nonprotein antigens remain largely underrepresented in our understanding of the molecular repertoire of innate and adaptive immunity. One such antibody is Mannitou, a murine IgM that recognizes paucimannosidic glycans. In this work, we report the production and purification of the recombinant antigen-binding fragment (Fab) of Mannitou IgM (Mannitou Fab) and employ a combination of biochemical and biophysical approaches to obtain its initial structural characterization. To this end, recombinant Mannitou Fab comprising the light chain (VL-CL) and heavy chain (VH-Cμ1) was produced in HEK293 FreeStyle cells and purified by cobalt-affinity chromatography followed by size-exclusion chromatography (SEC), which revealed two distinct oligomeric states consistent with a predominant monomeric form and a minor dimeric form. We employed SEC inline with multi-angle light scattering (SEC-MALS) and SEC coupled to small-angle X-ray scattering (SEC-SAXS) to establish that Mannitou Fab indeed adopts monomeric and dimeric forms in solution. Interestingly, Mannitou Fab is N-glycosylated at Asn164 of the heavy chain via HexNAc(5)Hex(6)Fuc(1–3) as revealed by mass spectrometry. We leveraged this information in conjunction with predicted structures of Mannitou Fab to facilitate the interpretation and modelling of SAXS data, leading to a plausible model for glycosylated Mannitou Fab. Analysis of the two chromatographically isolatable forms of Mannitou Fab using synchrotron-radiation circular dichroism revealed that the heat-denaturated Mannitou Fab monomer shares similar secondary-structural elements with the Mannitou Fab dimer, indicating that the latter may be misfolded. Collectively, the findings of this study will set the stage for future structural studies of Mannitou Fab and contribute to our understanding of possible side products due to misfolding during the production of recombinant Fabs, highlighting the importance of glycosylation in obtaining stable and monodisperse monomeric forms of recombinant Fabs.

α-Synuclein (aSyn) aggregation represents a key event in the neurodegenerative cascade of synucleinopathies. Initially, aSyn appears as an intrinsically disordered protein. However, its structural flexibility allows aSyn to either adopt α-helical conformations, relevant for physiological functions at presynaptic vesicles, or form β-strand-rich aggregates, leading to toxic oligomers. This relation between structure, function, and toxicity can be influenced by post-translational modifications such as the recently identified glutaminyl cyclase-catalyzed pyroglutamate (pE) modification. Here, we investigated (i) structural characteristics of monomeric, dimeric, and oligomeric states of N-terminal truncated, pE-modified aSyn variants, pE24-, pE62-, and pE79-aSyn by a complementary biophysical approach including DLS, SEC-MALS, SRCD, SEC-SAXS, and AUC and (ii) the toxicity of oligomeric pE-aSyn variants compared to full-length aSyn. Overall, pE62-aSyn showed an immediate fibril formation, reflecting the aggregation-prone properties of this particular variant. Furthermore, in a membrane-like environment, the secondary aSyn structure shifted toward α-helical folding depending on the degree of N-terminal truncation. pE79-aSyn showed a significantly reduced level of structural adaptation, reflecting compromised functions at presynaptic vesicles. In addition, the comparative analysis indicates the presence of a dimeric aSyn intermediate, the initial and potentially crucial step in aSyn aggregation, and supports the hypothesis of a toxic porous oligomeric state. For the first time, based on SAXS data, EOM models of the dimeric aSyn state are proposed.

Enhancing the local substrate concentration is a crucial strategy in nature for facilitating the proximity of two enzymes. The substrate of the first enzyme is transformed into a by-product that travels to the active site of the second enzyme without external diffusion, then transformed into a product and eventually expelled from the complex. In an effort to optimize the antimicrobial properties of myeloperoxidase from Rhodopirellula baltica (RbMPO), we created a library of fused chimeras between a glucose oxidase (GOx) and RbMPO so that H2O2 could be continuously perfused in the vicinity RbMPO, enabling the production of HOCl or HOSCN, well-known antimicrobial agents. The enzymes were characterized biochemically, enzymatically, and physically using low-resolution techniques such as AFM, SAXS, and cryofracture. SAXS experiments revealed that the chimeras were properly folded and existed in different oligomeric states. The kinetic parameters of the chimeras were determined and used for classification, revealing that all chimeras exhibited varying levels of activity and were microbicidal. The mixture of different oligomeric states of LEGGEAEA displayed both activity and microbicidal properties. AFM was used to visualize the chimeras in different oligomeric states, with their overall shapes ranging from round, oblong, to hooked, depending on the linker used.

Antimicrobial peptides (AMPs) are seen as a promising alternative to conventional antibiotics, yet their mechanism of action is not fully understood. We recently proposed that AMPs do not act as isolated monomers but instead aggregate into nano-sized clusters before targeting the membrane. However, cationic peptides may struggle to form clusters large enough to achieve effective membrane disruption alone, suggesting the involvement of a co-actor in this process. Here, we tackle this issue through both in silico and in vitro studies of mono- and bicomponent solutions comprising two newly identified peptides, one charged and the other neutral, before the interaction with the target membrane. We observe that while the charged peptides alone do not form clusters of noticeable size, in the mixture with the neutral peptides they coalesce into layered aggregates with a hydrophobic core of the neutral moiety and the charged monomers exposed to the surrounding solvent. The size of these mixed charged/neutral peptides aggregates is smaller than the ones in the neutral monocomponent solution due to the counteraction of electrostatic repulsion to the hydrophobic effect. We regard the structures thus formed as an ideal transport system - locking hydrophobic uncharged residues in the cluster core prevents interaction with eukaryotic membranes, while solvent-exposed charged residues facilitate electrostatic interaction with bacterial surfaces.

Homologous recombination (HR) is essential for the repair of DNA double-strand breaks and the restart of stalled replication forks. A critical step in HR is the formation of Rad51 nucleofilaments, which perform homology search and strand invasion of a homologous DNA sequence required for repair synthesis. In the yeast Saccharomyces cerevisiae, Rad52 facilitates Rad51 nucleofilament formation by mediating Rad51 loading onto ssDNA and counteracting Rad51 filament dissociation by the DNA translocase Srs2. The molecular basis of these two Rad52 functions remains unclear. Our integrative structural analyses of the Rad51-Rad52 interaction, combining NMR, SAXS, and modeling, reveal that an 85-residue segment of Rad52, conserved in fungi, folds upon binding to a broad surface of a Rad51 monomer. Notably, it includes an FxxA motif conserved in the BRC repeats of BRCA2 and at the Rad51-Rad51 interface. This binding mode was validated through an extensive set of mutations. Using in vivo assays and a functional fluorescent GFP-Rad51 fusion protein, we demonstrated that this entire segment is critical for Rad51 filament formation. These findings highlight how Rad52 functions as an assembly chaperone by preventing Rad51 oligomerization, promoting nucleation of Rad51 nucleofilaments on ssDNA, and counteracting the effects of Srs2 on destabilizing Rad51 filaments.

Small-angle x-ray scattering (SAXS) has evolved over the past decades into an essential structural biology technique for the study of conformational states of solubilized biomacromolecules and assemblies. It complements a multitude of other methods, including the atomic-resolution techniques NMR, crystallography, and cryo-EM. In the vast majority of applications, the biological systems of interest are measured by SAXS in aqueous buffers. This approach, although quick and straightforward, limits the information content of the internal topology of biomacromolecular assemblies of variable electron density, such as protein-RNA/DNA complexes, and detergent/lipid-solubilized membrane proteins. These shortcomings can be partially overcome by altering the solvent electron density by the addition of small, electron-rich molecules such as sugar, glycerol, salt, and heavy-atom compounds, so-called contrast agents. A crucial issue in these contrast-variation SAXS experiments is the question whether the conformational state and the enzymatic activity of biomolecules are maintained after long incubation times with contrast agents, which are often required at molar concentrations. Here, we present a structural SAXS study of two proteins, a bacterial endo-β-1,4-mannanase and a malate dehydrogenase, in the presence of high concentrations of sucrose and a lanthanide compound and evaluate the residual enzymatic activity as a function of the incubation time. Our results indicate that the global conformation and the oligomeric state of both proteins are preserved over the entire concentration range of both contrast agents, and that their enzymatic activities are unaltered after long incubation times (up to 24 h). Taken together, our results encourage a more routine use, as well as further developments, of contrast variation SAXS experiments for structural studies of a variety of important and challenging biomacromolecular assemblies.

Engineered protein cages are of great interest considering their diverse applications in delivery and catalysis. Here, we describe macrocycle-triggered icosahedral cage assembly of a designed beta-propeller. Cage assembly was evidenced by small-angle X-ray scattering and X-ray crystallography.

The red macroalgae Porphyra , commonly known as Nori, is widely used as food around the world due to its high nutrient content, including the significant abundance of colored phycobiliproteins (PBPs). Among these, R-phycocyanin (R-PC) stands out for its vibrant purple color and numerous bioactive properties, making it a valuable protein for the food industry. However, R-PC's limited thermal stability necessitates alternative processing methods to preserve its color and bioactive properties. Our study aimed to investigate the in-situ stability of oligomeric R-PC under high pressure (HP) conditions (up to 4000 bar) using a combination of absorption, fluorescence, and small-angle X-ray scattering (SAXS) techniques. The unfolding of R-PC is a multiphase process. Initially, low pressure induces conformational changes in the R-PC oligomeric form (trimers). As pressure increases above 1600 bar, these trimers dissociate into monomers, and at pressures above 3000 bar, the subunits begin to unfold. When returned to atmospheric pressure, R-PC partially refolds, retaining 50% of its original color absorbance. In contrast, heat treatment causes irreversible and detrimental effects on R-PC color, highlighting the advantages of HP treatment in preserving both the color and bioactive properties of R-PC compared to heat treatment.

Supramolecular synthons, defined as reproducible intermolecular structural units, have greatly aided small molecule crystal engineering. In this paper, we propose that supramolecular synthons guide ligand-mediated protein crystallization. The protein RSL and the macrocycle sulfonato-calix[8]arene cocrystallize in at least four ways. One of these cocrystals is a highly porous cube comprising protein nodes connected by calixarene dimers. We show that mutating an aspartic acid to an asparagine results in two new cubic assemblies that depend also on the crystallization method. One of the new cubic arrangements is mediated by calixarene trimers and has a ∼30% increased cell volume relative to the original crystal with calixarene dimers. Crystals of the sulfonato-calix[8]arene sodium salt were obtained from buffered conditions similar to those used to grow the protein-calix[8]arene cocrystals. X-ray analysis reveals a coordination polymer of the anionic calix[8]arene and sodium cation in which the macrocycle is arranged as staggered stacks of the pleated loop conformation. Remarkably, the calixarene packing arrangement is the same in the simple salt as in the protein cocrystal. With the pleated loop conformation, the calixarene presents an extended surface for binding other calixarenes (oligomerization) as well as binding to a protein patch (biomolecular complexation). Small-angle X-ray scattering data suggest pH-dependent calixarene assembly in solution. Therefore, the calix[8]arene-calix[8]arene structural unit may be regarded as a supramolecular synthon that directs at least two types of protein assembly, suggesting applications in protein crystal engineering.

Eukaryotic innate immune systems use pattern recognition receptors to sense infection by detecting pathogen-associated molecular patterns, which then triggers an immune response. Bacteria have similarly evolved immunity proteins that sense certain components of their viral predators, known as bacteriophages1-6. Although different immunity proteins can recognize different phage-encoded triggers, individual bacterial immunity proteins have been found to sense only a single trigger during infection, suggesting a one-to-one relationship between bacterial pattern recognition receptors and their ligands7-11. Here we demonstrate that the antiphage defence protein CapRelSJ46 in Escherichia coli can directly bind and sense two completely unrelated and structurally different proteins using the same sensory domain, with overlapping but distinct interfaces. Our results highlight the notable versatility of an immune sensory domain, which may be a common property of antiphage defence systems that enables them to keep pace with their rapidly evolving viral predators. We found that Bas11 phages harbour both trigger proteins that are sensed by CapRelSJ46 during infection, and we demonstrate that such phages can fully evade CapRelSJ46 defence only when both triggers are mutated. Our work shows how a bacterial immune system that senses more than one trigger can help prevent phages from easily escaping detection, and it may allow the detection of a broader range of phages. More generally, our findings illustrate unexpected multifactorial sensing by bacterial defence systems and complex coevolutionary relationships between them and their phage-encoded triggers.

The soluble glucose dehydrogenase (sGDH) from Acinetobacter calcoaceticus has been widely studied and is used, in biosensors, to detect the presence of glucose, taking advantage of its high turnover and insensitivity to molecular oxygen. This approach, however, presents two drawbacks: the enzyme has broad substrate specificity (leading to imprecise blood glucose measurements) and shows instability over time (inferior to other oxidizing glucose enzymes). We report the characterization of two sGDH mutants: the single mutant Y343F and the double mutant D143E/Y343F. The mutants present enzyme selectivity and specificity of 1.2 (Y343F) and 5.7 (D143E/Y343F) times higher for glucose compared with that of the wild-type. Crystallographic experiments, designed to characterize these mutants, surprisingly revealed that the prosthetic group PQQ (pyrroloquinoline quinone), essential for the enzymatic activity, is in a cleaved form for both wild-type and mutant structures. We provide evidence suggesting that the sGDH produces H2O2, the level of production depending on the mutation. In addition, spectroscopic experiments allowed us to follow the self-degradation of the prosthetic group and the disappearance of sGDH's glucose oxidation activity. These studies suggest that the enzyme is sensitive to its self-production of H2O2. We show that the premature aging of sGDH can be slowed down by adding catalase to consume the H2O2 produced, allowing the design of a more stable biosensor over time. Our research opens questions about the mechanism of H2O2 production and the physiological role of this activity by sGDH.

d-Amino acid residues, found in countless peptides and natural products including ribosomally synthesized and post-translationally modified peptides (RiPPs), are critical for the bioactivity of several antibiotics and toxins. Recently, radical S-adenosyl-l-methionine (SAM) enzymes have emerged as the only biocatalysts capable of installing direct and irreversible epimerization in RiPPs. However, the mechanism underpinning this biochemical process is ill-understood and the structural basis for this post-translational modification remains unknown. Here we report an atomic-resolution crystal structure of a RiPP-modifying radical SAM enzyme in complex with its substrate properly positioned in the active site. Crystallographic snapshots, size-exclusion chromatography–small-angle x-ray scattering, electron paramagnetic resonance spectroscopy and biochemical analyses reveal how epimerizations are installed in RiPPs and support an unprecedented enzyme mechanism for peptide epimerization. Collectively, our study brings unique perspectives on how radical SAM enzymes interact with RiPPs and catalyze post-translational modifications in natural products. Peptide epimerization is a common but enigmatic post-translational modification found in antibiotics formed from ribosomally synthesized and post-translationally modified peptides. Now, crystallographic snapshots, spectroscopy and biochemical investigations have provided insight into the mechanism of peptide epimerization catalyzed by radical S-adenosyl-l-methionine epimerases.

Homologous recombination (HR) is essential for the repair of DNA double-strand breaks and the restart of stalled replication forks. A critical step in HR is the formation of Rad51 nucleofilaments, which perform homology search and strand invasion of a homologous DNA sequence required for repair synthesis. In the yeast Saccharomyces cerevisiae, Rad52 facilitates Rad51 nucleofilament formation by mediating Rad51 loading onto ssDNA and counteracting the dissociation of Rad51 filaments by the DNA translocase Srs2. The molecular basis of these two Rad52 functions remains unclear. Our integrative structural analyses of the Rad51-Rad52 interaction, combining NMR, SAXS, and modeling, reveal that an 85-residue segment of Rad52, conserved in fungi, folds upon binding to a broad surface of a Rad51 monomer. Notably, it includes an FxxA motif conserved in the BRC repeats of BRCA2 and at the Rad51-Rad51 interface. This binding mode was validated through an extensive set of mutations. Using in vivo assays and a functional fluorescent GFP-Rad51 fusion protein, we demonstrated that this entire segment is critical for Rad51 filament formation. These findings highlight how Rad52 functions as an assembly chaperone, preventing Rad51 oligomerization, promoting nucleation of Rad51 nucleofilaments on ssDNA, and protecting Rad51 filaments from destabilization by Srs2. ### Competing Interest Statement The authors have declared no competing interest.

Lung cancer cells resistant to radiotherapy present a significant clinical challenge. Stable telomeric structures, maintained by the TRF2 protein, play a critical role in protecting cells from ionizing radiation. Reduced TRF2 expression increases DNA damage and radiosensitivity. We designed a self-assembling system utilizing ultra-small luminescent gold nanoclusters (AuNCs) with radiosensitizing properties, combined with siRNA targeting TRF2. The system forms ≈100  nm non-spherical structures with AuNCs enriched in the outer layer, exhibiting a 17.6-fold enhancement in red photoluminescence due to aggregation-induced effects. This nanoplatform efficiently penetrates lung cancer cells, reducing TRF2 expression by 50%. Under 5 Gy radiotherapy, cells treated with this system show a 1.5-fold radiosensitivity increase from AuNCs and a 2.3-fold reduction in clonogenic survival due to telomere deprotection. The AuNC-siRNATRF2 system combines enhanced optical properties with biological functionality, offering a promising approach to augment radiotherapy efficacy by disrupting telomeric protective mechanisms in cancer cells.

Genome and epigenome integrity in eukaryotes depends on the proper coupling of histone deposition with DNA synthesis. This process relies on the evolutionary conserved histone chaperone CAF-1 for which the links between structure and functions are still a puzzle. While studies of the Saccharomyces cerevisiae CAF-1 complex enabled to propose a model for the histone deposition mechanism, we still lack a framework to demonstrate its generality and in particular, how its interaction with the polymerase accessory factor PCNA is operating. Here, we reconstituted a complete SpCAF-1 from fission yeast. We characterized its dynamic structure using NMR, SAXS and molecular modeling together with in vitro and in vivo functional studies on rationally designed interaction mutants. Importantly, we identify the unfolded nature of the acidic domain which folds up when binding to histones. We also show how the long KER helix mediates DNA binding and stimulates SpCAF-1 association with PCNA. Our study highlights how the organization of CAF-1 comprising both disordered regions and folded modules enables the dynamics of multiple interactions to promote synthesis-coupled histone deposition essential for its DNA replication, heterochromatin maintenance, and genome stability functions.

Translationally Controlled Tumor Protein (TCTP) serves as a pro-survival factor in tumor cells, inhibiting the mitochondrial apoptosis pathway by enhancing the function of antiapoptotic Bcl-2 family members Mcl-1 and Bcl-xL. TCTP specifically binds to Bcl-xL, preventing Bax-dependent Bcl-xLinduced cytochrome c release, and it reduces Mcl-1 turnover by inhibiting its ubiquitination, thereby decreasing Mcl-1mediated apoptosis. TCTP harbors a BH3-like motif that forms a beta-strand buried in the globular domain of the protein. In contrast, the crystal structure of the TCTP BH3-like peptide in complex with the Bcl-2 family member Bcl-xL reveals an alpha-helical conformation for the BH3-like motif, suggesting significant structural changes upon complex formation. Employing biochemical and biophysical methods, including limited proteolysis, circular dichroism, NMR, and SAXS, we describe the TCTP complex with the Bcl-2 homolog Mcl-1. Our findings demonstrate that full-length TCTP binds to the BH3 binding groove of Mcl-1 via its BH3-like motif, experiencing conformational exchange at the interface on a micro- to milli-second timescale. Concurrently, the TCTP globular domain becomes destabilized, transitioning into a molten-globule state. Furthermore, we establish that the non-canonical residue D16 within the TCTP BH3-like motif reduces stability while enhancing the dynamics of the intermolecular interface. In conclusion, we detail the structural plasticity of TCTP and discuss its implications for partner interactions and future complexes.