Biology subjects

Legrand, P.

Publications and source records attributed to Legrand, P..

17 recordsLinked to original sources

bioRxiv2026

BRCA2 and RAD54B FxPP motifs Bind DMC1 Filaments through Persistent and Transient Interfaces

During meiosis, double-strand DNA breaks are programmed to initiate homologous recombination. The DMC1 recombinogenic filament is central to the repair of these breaks. However, the three-dimensional structures of the protein-protein interfaces regulating the assembly and activity of this filament have not been described yet. We and others previously reported that in BRCA2, a P-motif called PhePP binds to DMC1 in its oligomeric and filament states. Here we identified a similar P-motif in the DNA translocase RAD54B. We solved the cryo-electron microscopy (cryo-EM) structures of BRCA2 and RAD54B P-motif peptides bound to a ssDNA-DMC1 filament at 1.9-2.0 [A] resolution. While these peptides only share the sequence F-[IV]-P-P, they bind to the filament through larger 9-10 residue core sequences with superimposable structures. Both peptides bind to a hydrophobic and negatively charged site, named the P-site, on a single DMC1 protomer and stabilize the ssDNA-DMC1 filament. Mutagenesis experiments and molecular dynamics simulations identified additional transient interactions between positively charged residues of the peptides and negatively charged patches distributed on the DMC1 protomers. We propose that BRCA2 and RAD54B stably dock at the P-site of a single DMC1 protomer but also bridge two DMC1 protomers within the presynaptic filament via transient contacts with the adjacent protomer.

biochemistry↗

bioRxiv2026

Structural basis for continuous DNA-end protection during ligation of double-strand breaks in yeast Non-Homologous End-Joining

Non-homologous end joining (NHEJ) repairs DNA double-strand breaks by synapsing and ligating DNA ends. In vertebrates, DNA-PKcs promotes end alignment and processing, yet several eukaryotes - including budding yeast - perform NHEJ without DNA-PKcs, and the underlying mechanism remains unclear. Here we report cryo-electron microscopy structures of reconstituted Saccharomyces cerevisiae NHEJ synaptic complexes assembled on DNA ends bearing either 4-bp terminal microhomologies or blunt termini. On the microhomology substrate with a single 5-P, we capture a ligation-competent short-range complex in which a single Dnl4 catalytic core is fully ordered and engaged at the 5'-adenylated nick. When two 5-P are present, we resolve two predominant, coexisting DNA-aligned protective states in which both Dnl4 DBD-NTD modules occupy the break region with reciprocal geometries, supporting an alternating engagement model for sequential sealing of the two strands while maintaining continuous end association. In contrast, blunt-ended substrates yield a non-aligned protective configuration in which two Dnl4 catalytic modules constrain the DNA ends [~]30 [A] apart in a ligation-incompatible arrangement, providing a structural explanation for slow blunt-end joining in yeast. Together, these structures define architectural and mechanistic principles of DNA-PKcs-independent NHEJ.

molecular biology↗

bioRxiv2025

Quantitative mapping of methionine sensitivity to oxidation in the copper-bound PcuC chaperone

Copper is typically coordinated by histidine, cysteine, or methionine in proteins, and these residues are particularly sensitive to oxidation. However, it remains unclear whether copper-coordinating residues are more prone to oxidation than non-coordinating ones, and how their susceptibility changes between the apo and copper-bound states. The copper chaperone PcuC, important for cytochrome c oxidase assembly in bacteria, contains a canonical binding site composed of two histidines and two methionines (H51xnM63x22H86xM88), as well as a disordered C-terminal extension enriched in methionine and histidine. To quantify methionine oxidation sensitivity in both apo- and Cu-bound PcuC, we used a methionine-specific oxaziridine probe combined with mass spectrometry and compared labeling patterns to those generated by 18O-labeled hydrogen peroxide. We show that methionine residues display distinct oxidation sensitivities in the apoprotein, and that the oxaziridine reacts similarly to H218O2. Importantly, this probe enables quantification of methionine oxidation independently of hydroxyl radicals generated by copper-driven Fenton chemistry, which lacks residue specificity. In the copper-bound form, Cu binding strongly alters methionine reactivity, with a marked increase in oxidation of the coordinating Met63 and Met88. Structural analysis revealed that two copper ions occupy the canonical site, while the C-terminal extension does not contribute to coordination. Comparison of structural features and oxidation values showed that methionine sensitivity correlates with solvent exposure in the folded domain, but with local positive charge in the disordered region. These findings demonstrate that copper coordination modulates methionine oxidation, and that oxaziridine-based probes provide powerful tools for mapping oxidation sensitivity in (metallo)proteins.

biochemistry↗

bioRxiv2025

Subcellular reorganization upon phage infection reveals stepwise assembly of viral particles from membrane-associated precursors

Summary paragraphViruses are obligate intracellular parasites and viral infections lead to massive host cell rearrangement to support the rapid generation of progeny. Host take-over and remodelling include formation of viral-induced compartments for viral genome replication and/or assembly. While viruses infecting bacteria, bacteriophages or phages, have been extensively characterized in vitro, the molecular mechanisms underlying the viral cycle inside the crowded cytoplasm remain unclear. Here, we investigate the spatial reorganization of SPP1-infected bacteria under near-native conditions by electron cryo tomography. The most prominent feature is the formation of a large viral DNA (vDNA) compartment from which ribosomes are excluded. In SPP1 infection, there is no membrane nor proteinaceous shell surrounding these compartments. Also, we identified novel key intermediates in virus assembly: open precursors of procapsid lattice were found at the cytoplasmic membrane in a process that requires expression of the portal protein. Next, DNA-free procapsids relocate inside the vDNA compartment where vDNA is packed in a stepwise manner. Finally, DNA-filled capsids segregate to the periphery of the compartment for assembly completion and storage. Collectively, we provide comprehensive mechanistic insights into the complete viral assembly pathway of SPP1 directly in cellula and show how specific steps are coordinated inside the reorganized bacterial cell.

cell biology↗

bioRxiv2025

Molecular determinants of species-specific cell-cell recognition activating the class II gamete fusogen HAP2

Species-specific adhesion of complementary gametes is a prerequisite to cell fusion during fertilization. Despite its importance, the molecular mechanism underlying cell-cell recognition and membrane adhesion to drive gamete fusion is not fully understood for any organism. In the alga, Chlamydomonas, the species-specific gamete adhesion protein MAR1 guides the fusogen HAP2 into a pre-fusion conformation on mt- gametes and serves as a receptor for the FUS1 adhesion protein on mt+ gametes. Here, we show that soluble recombinant MAR1 completely blocks the HAP2-dependent gamete fusion process by binding to FUS1 on mt+ gametes and report the X-ray structure of the MAR1-FUS1 complex. In vivo gamete fusion experiments showed that mutations of key residues at the observed MAR1-FUS1 interface strongly impair adhesion, yet gamete fusion still proceeds, highlighting the efficiency of the fusion-activation process. These findings uncover the molecular architecture of a surface receptor complex whose formation releases the viral class II viral fusion-protein ortholog HAP2, allowing it to undergo its membrane-fusogenic conformational change.

cell biology↗

bioRxiv2024

Communication between DNA polymerases and Replication Protein A within the archaeal replisome

Replication Protein A (RPA) plays a pivotal role in DNA replication by coating and protecting exposed single-stranded DNA, and acting as a molecular hub that recruits additional replication factors. We demonstrated that archaeal RPA hosts a winged-helix domain (WH) that interacts with two key actors of the replisome: the DNA primase (PriSL) and the replicative DNA polymerase (PolD). Using an integrative structural biology approach, combining nuclear magnetic resonance, X-ray crystallography and cryo-electron microscopy, we unveiled how RPA interacts with PriSL and PolD through two distinct surfaces of the WH domain: an evolutionarily conserved interface and a novel binding site. Finally, RPA was shown to stimulate the activity of PriSL in a WH-dependent manner. This study provides a molecular understanding of the WH-mediated regulatory activity in conserved central replication factors such as RPA, which regulate genome maintenance in Archaea and Eukaryotes.

molecular biology↗

bioRxiv2024

Arf family GTPases are present in Asgard archaea

The emergence of eukaryotes from their prokaryotic ancestors is one of the most fundamental evolutionary events in the history of life. Little is robustly known about how eukaryogenesis occurred, but a major breakthrough came with the identification of the Asgardarchaeota, the closest prokaryotic lineage to eukaryotes yet discovered. Endomembrane organelles, and the capacity to transport material between them, are major hallmarks of eukaryotic cells. The Arf family GTPases are crucial regulators of organelle dynamics in eukaryotes, functioning in vesicle budding, membrane tethering and membrane-cytoskeleton interactions. Although an expanded GTPase complement has been reported in the Asgardarchaeota, the specific origins of the Arf family remain elusive. Here we report a new group of prokaryotic GTPases, the ArfRs. Widely present in Asgardarchaeota and almost exclusive to them, it is the clade from which all eukaryotic Arf family proteins are derived. Heterologous expression of representative Asgardarchaeota ArfR proteins in the model eukaryote Saccharomyces cerevisiae and X-ray crystallographic studies demonstrate that ArfR GTPases possess the mechanism of membrane binding and structural features unique to Arf family proteins. Our results show that Arf family GTPases are present in Asgardarchaeota, and strongly suggest that they originated in the archaeal contributor to eukaryogenesis, providing support for nascent endomembrane system capacity evolving early in eukaryogenesis.

evolutionary biology↗

bioRxiv2023

DMC1 and RAD51 bind FxxA and FxPP motifs of BRCA2 via two separate interfaces

In vertebrates, the BRCA2 protein is essential for meiotic and somatic homologous recombination (HR) due to its interaction with RAD51 and DMC1 strand exchange proteins (recombinases). The interaction is mediated by FxxA and FxPP motifs, whose defining feature is the invariant phenylalanine. The FxxA motifs, present in the eight BRC repeats in the central region of BRCA2, compete with the FxxA motif of the linker region of RAD51 that is responsible for recombinase self-oligomerization. In vitro, BRCs disrupt RAD51 nucleoprotein filaments, but they are essential for RAD51 function in the context of the full-length BRCA2 protein. The role of the FxPP motifs is poorly studied but they also contribute to BRCA2 function in cells. In particular, the C-terminal TR2/CTRB domain of BRCA2, which contains an FxPP motif, is required for stabilization of RAD51 filament and replication fork protection. We recently found that deletion of the BRCA2 PhePP domain, which contains another FxPP motif, disrupts DMC1 but not RAD51 function in meiosis. Here we provide a mechanistic explanation for this phenotype by solving the crystal structure of the complex between DMC1 and the PhePP domain of BRCA2. Our structure reveals that, despite sequence similarity, the A-motifs (FxxA) and P-motifs (FxPP) bind to distinct and contiguous sites on the recombinases. The PhePP P-motif binding site is mostly located at the ATPase domain surface of a DMC1 monomer, but also extends to the linker region of the adjacent monomer, thus engaging two adjacent protomers in the DMC1 oligomer. Our structural analysis provides a mechanism explaining how PhePP favors the formation of the DMC1 nucleoprotein filament and stabilizes it. It corroborates and explains the stabilizing effect of the P-motif from BRCA2 TR2/CTRB on RAD51.

biochemistry↗

bioRxiv2023

Disordered regions and folded modules in CAF-1 promote histone deposition in S. pombe

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 S. 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.

biochemistry↗

bioRxiv2023

BRCA2-HSF2BP Oligomeric Ring Disassembly by BRME1 Promotes Homologous Recombination

In meiotic homologous recombination (HR), BRCA2 facilitates loading of the recombinases RAD51 and DMC1 at the sites of double-strand breaks. The HSF2BP-BRME1 complex interacts with BRCA2 to support its function in meiotic HR. In somatic cancer cells ectopically producing HSF2BP, DNA damage can trigger HSF2BP-dependent degradation of BRCA2, which prevents HR. Here we show that, upon binding to BRCA2, HSF2BP assembles into a large ring-shaped 24-mer consisting of three interlocked octameric rings. Addition of BRME1 leads to dissociation of this ring structure, and cancels the disruptive effect of HSF2BP on cancer cell resistance to DNA damage. It also prevents BRCA2 degradation during inter-strand DNA crosslink repair in Xenopus egg extracts. We propose that the control of HSF2BP-BRCA2 oligomerization by BRME1 ensures timely assembly of the ring complex that concentrates BRCA2 and controls its turnover, thus promoting meiotic HR.

biochemistry↗

bioRxiv2022

Molecular interactions of PCSK9 with an inhibitory nanobody, CAP1 and HLA-C: functional regulation of LDLR levels

ObjectiveThe liver-derived circulating PCSK9 enhances the degradation of the LDL receptor (LDLR) in endosomes/lysosomes. PCSK9 inhibition or silencing is presently used in clinics worldwide to reduce LDL-cholesterol, resulting in lower incidence of cardiovascular disease and possibly cancer/metastasis. The mechanism by which the PCSK9-LDLR complex is sorted to degradation compartments is not fully understood. We previously suggested that out of the three M1, M2 and M3 subdomains of the C-terminal Cys/His-rich-domain (CHRD) of PCSK9, only M2 is critical for the activity of extracellular of PCSK9 on cell surface LDLR. This likely implicates the binding of M2 to an unknown membrane-associated "protein X" that would escort the complex to endosomes/lysosomes for degradation. We reported that a nanobody P1.40 binds the M1 and M3 domains of the CHRD and inhibits the function of PCSK9. It was also reported that the cytosolic adenylyl cyclase-associated protein 1 (CAP1) could bind M1 and M3 subdomains and enhance the activity of PCSK9. In this study, we determined the 3-dimensional structure of the CHRD-P1.40 complex to understand the intricate interplay between P1.40, CAP1 and PCSK9 and how they regulate LDLR degradation. MethodsX-ray diffraction of the CHRD-P1.40 complex was analyzed with a 2.2 [A] resolution. The affinity and interaction of PCSK9 or CHRD with P1.40 or CAP1 was analyzed by atomic modeling, site-directed mutagenesis, bio-layer interferometry, expression in hepatic cell lines and immunocytochemistry to monitor LDLR degradation. The CHRD-P1.40 interaction was further analyzed by deep mutational scanning and binding assays to validate the role of predicted critical residues. Conformational changes and atomic models were obtained by small angle X-ray scattering (SAXS). ResultsWe demonstrate that PCSK9 exists in a closed or open conformation and that P1.40 favors the latter by binding key residues in the M1 and M3 subdomains of the CHRD. Our data show that CAP1 is well secreted by hepatic cells and binds extracellular PCSK9 at distinct residues in the M1 and M3 domains and in the acidic prodomain. CAP1 stabilizes the closed conformation of PCSK9 and prevents P1.40 binding. However, CAP1 siRNA only partially inhibited PCSK9 activity on the LDLR. By modeling the previously reported interaction between M2 and an R-X-E motif in HLA-C, we identified Glu567 and Arg549 as the critical M2 residues binding HLA-C. Amazingly, these two residues are also required for the PCSK9-induced LDLR degradation. ConclusionsThe present study reveals that CAP1 enhances the function of PCSK9, likely by twisting the protein into a closed configuration that exposes the M2 subdomain needed for targeting the PCSK9-LDLR complex to degradation compartments. We hypothesize that "protein X", which is expected to guide the LDLR-PCSK9-CAP1 complex to these compartments after endocytosis into clathrin-coated vesicles, is HLA-C or a similar MHC-I family member. This conclusion is supported by the PCSK9 natural loss-of-function Q554E and gain-of-function H553R M2 variants, whose consequences are anticipated by our modeling.

molecular biology↗

bioRxiv2022

DNA-binding mechanism and evolution of Replication Protein A

Replication Protein A (RPA) is a heterotrimeric single stranded DNA-binding protein with essential roles in DNA replication, recombination and repair, in both eukaryotic and archaeal cells. By using an integrative approach that combines three crystal structures, four cryo-EM structures in complex with single-stranded DNA (ssDNA) of different lengths, we extensively characterized RPA from Pyrococcus abyssi in different states. These structures show two essential features conserved in eukaryotes: a trimeric core and a module that promotes cooperative binding to ssDNA, as well as a newly identified archaeal-specific domain. These structures reveal for the first time how ssDNA is handed over from one RPA complex to the other, and uncover an unanticipated mechanism of self-association on ssDNA tracts. This work constitutes a significant step forward in the molecular understanding of the structure and DNA-binding mechanism of RPA, with far-reaching implications for the evolution of this primordial replication factor in Archaea and Eukarya.

biochemistry↗

bioRxiv2022

The LH-DH module of the bacterial replicative helicases is the common binding site for DciA and other helicase loaders

During the initiation step of bacterial genome replication, replicative helicases depend on specialized proteins for their loading onto oriC. DnaC and DnaI were the first loaders characterized. However, most bacteria do not contain any of these genes, which are domesticated phage elements that replaced the ancestral and unrelated loader gene dciA several times during evolution. To understand how DciA assists the loading of DnaB, we determined the crystal structure of the complex from Vibrio cholerae, in which two VcDciAs interact with a dimer of VcDnaB, without changing its canonical structure. Our data showed that the VcDciA binding site on VcDnaB is the conserved module formed by the linker helix LH of one monomer and the determinant helix DH of the second one. Interestingly, DnaC from Escherichia coli also targets this module onto EcDnaB. Thanks to their common target site, we showed that VcDciA and EcDnaC could be functionally interchanged in vitro, despite sharing no structural similarities. This is a milestone in understanding the mechanism employed by phage helicase loaders to hijack bacterial replicative helicases during evolution.

molecular biology↗

bioRxiv2022

The BAF A12T mutation reduces BAF affinity to lamin A/C, preventing its recruitment to nuclear ruptures in Nestor Guillermo Progeria Syndrome cells.

The premature aging disorder Nestor Guillermo Progeria Syndrome (NGPS) is caused by a homozygous Alanine to Threonine mutation at position 12 (A12T) in Barrier-to- Autointegration Factor (BAF). BAF is a small essential protein that binds to DNA and nuclear envelope proteins. It contributes to important cellular processes including transcription regulation and nuclear envelope reformation after mitosis. More recently, BAF was identified as an important factor for nuclear envelope repair upon rupture in interphase. However, the mechanism by which the BAF A12T mutation causes NGPS has remained unclear. To investigate the effects of this mutation on nuclear envelope integrity, we used NGPS-derived patient cells and engineered an isogenic cell line by reversing the BAF A12T homozygous mutation using CRISPR/Cas9. Using a combination of cellular models, structural data and in vitro assays, we identified that the A12T mutation reduces the affinity of BAF for lamin A/C by tenfold. As a result, BAF A12T is unable to recruit lamin A/C to sites of nuclear envelope rupture. This leads to persistent lamin A/C gaps at sites of ruptures, and contributes to nuclear fragility in NGPS patient cells, which show increased frequency of nuclear envelope re- rupturing. Overexpression of wild-type BAF in a NGPS context rescues lamin A/C recruitment to sites of nuclear rupture, which could explain why the heterozygous A12T mutation does not cause premature aging.

cell biology↗

bioRxiv2021

ComF is a key mediator in single-stranded DNA transport and handling during natural transformation

Natural transformation plays a major role in the spreading of antibiotic resistances and virulence factors. Whilst bacterial species display specificities in the molecular machineries allowing transforming DNA capture and integration into their genome, the ComF(C) protein is essential for natural transformation in all Gram-positive and - negative species studied. Despite this, its role remains largely unknown. Here, we show that Helicobacter pylori ComF is not only involved in DNA transport through the cell membrane, but it also required for the handling of the ssDNA once it is delivered into the cytoplasm. ComF crystal structure revealed the presence of a zinc-finger motif and a putative phosphoribosyl transferase domain, both necessary for its in vivo activity. ComF is a membrane-associated protein with affinity for single-stranded DNA. Collectively, our results suggest that ComF provides the link between the transport of the transforming DNA into the cytoplasm and its handling by the recombination machinery.

microbiology↗

bioRxiv2020

A cryptic BRCA2 repeated motif binds to HSF2BP oligomers with no impact on meiotic recombination

BRCA2 plays a prominent role in meiotic homologous recombination (HR). Loss of BRCA2 or several of its meiotic partners causes fertility defects. One of these partners, HSF2BP, was recently discovered as expressed physiologically in germline and ectopically produced in cancer cells. It has an N-terminal coiled coil motif involved in direct binding to the protein BRME1, and both HSF2BP and BRME1 are essential for meiotic HR during spermatogenesis. It also interacts through its C-terminal Armadillo (ARM) domain with a conserved region of BRCA2 of unknown function. We analyzed the structural properties and functional consequences of the BRCA2-HSF2BP interaction and tested the emerging model of its involvement in meiosis. We solved the crystal structure of the complex between the BRCA2 fragment that is disordered in solution and the HSF2BP dimeric ARM domain. This revealed two previously unrecognized BRCA2 repeats that each interact with one ARM monomer from two different dimers. BRCA2 binding triggers ARM tetramerization, resulting in a complex containing two BRCA2 fragments connecting two ARM dimers. The 3D structures of the BRCA2 repeats are superimposable, revealing conserved contacts between the BRCA2 residues defining the repeats and the HSF2BP residues lining the groove of the ARM. This large interface is responsible for the nanomolar affinity of the interaction, significantly stronger than any other measured interaction involving BRCA2. Deleting exon 12 from Brca2, encoding the first repeat, disrupted BRCA2 binding to HSF2BP in vitro and in cells. However, Brca2{Delta}12/{Delta}12 mice with the same deletion were fertile and did not show any meiotic defects, contrary to the prediction from the model positing that HSF2BP acts as a meiotic localizer of BRCA2. We conclude that the high-affinity interaction between BRCA2 and HSF2BP and the resulting HSF2BP oligomerization are not required for RAD51 and DMC1 recombinase localization to meiotic double strand breaks and for productive meiotic HR.

molecular biology↗

bioRxiv2020

Downregulation of the essential Rrp44 ribonuclease causes extensive ultra-structure cell modifications in Trypanosoma brucei

Rrp44/Dis3 is an essential protein conserved in all eukaryotes that functions in the maturation of many different RNA precursors and RNA surveillance. The Trypanosoma brucei Rrp44/Dis3 homologue (TbRRP44) is required for maturation of pre-rRNA, spliced leader, and U3 snoRNA precursors. Its depletion leads to inhibition of cell proliferation and eventually to cell death. In this work, we showed that TbRRP44 depletion causes a massive expansion of acidic and lysosome-derived vacuoles, enlargement of cell and nuclei sizes without changes in DNA content, mitochondrial inactivation, and autophagy induction. Consistently, 3D reconstructions using cryo-soft X-ray tomography revealed extreme vacuolation of the cytoplasm and numerous cellular alterations, including an increase in size and number of calcium-containing vesicles and lipid droplets. These multiple defects indicate that a combination of alterations converge to induce lysosome expansion. With time, the cytoplasm is taken up by lysosome-derived vacuoles, which may be a final stage leading to the cell death triggered by TbRRP44 depletion. These studies provide the first evidence on the ultra-structure cell modifications caused by deficiency of this essential ribonuclease in T. brucei.

cell biology↗