I2BC Paris-Saclay

The Apicomplexa-specific ACDC domain adopts a never before seen protein fold.

In collaboration with the Nessler team at the I2BC and the Llinás lab at PSU, US, we reveal that the ACDC domain, only found in DNA-binding proteins of the Apicomplexa phylum, grouping several important human pathogens including malaria, has a never before seen protein fold. We also identify potential ligands that may be optimized in the future as protein inhibitors.

More information:https://journals.iucr.org/paper?S2059798324012518

Conatct: Joana SANTOS joana.santos@i2bc.paris-saclay.fr

We have identified a novel tubulin surface targeted by the Tau protein. From these results, we formulate new hypotheses on the regulation of microtubules by Tau and on Tau oligomerization.

Tau is a protein involved in the regulation of axonal microtubules in neurons. In pathological conditions, it forms filamentous aggregates which are molecular markers of the Alzheimer’s disease and of related neurodegenerative disorders collectively known as tauopathies. Structures of Tau in fibrils or bound to the microtubule have been reported. We have determined the structure of a Tau construct comprising the PHF6 motif, an hexapeptide involved in Tau aggregation, as a complex with tubulin. This Tau fragment binds as a dimer to a new site which, when transposed to the microtubule, would correspond to a pore between protofilaments. These results raise new hypotheses on Tau-induced microtubule assembly and stabilization and on Tau oligomerization. They reconcile apparently contradictory data from the literature, in particular concerning the coexistence of different binding modes of Tau to the microtubule.

More information : https://academic.oup.com/pnasnexus/article/3/11/pgae487/7850850

Contact : Benoît GIGANT benoit.gigant@i2bc.paris-saclay.fr

Following the French MOOC 'Voyage au cœur du vivant avec des rayons X : la cristallographie', available on FUN-MOOC from 2017 to 2022, and the book in French 'Introduction à la cristallographie biologique', we now present an English version.

Following the French MOOC 'Voyage au cœur du vivant avec des rayons X : la cristallographie', available on FUN-MOOC from 2017 to 2022, and the book in French 'Introduction à la cristallographie biologique', we now present an English version.
This book is primarily designed for beginners in biological crystallography.
It provides an introduction to the different steps of biological crystallography, from the crystallisation to the three-dimensional structure of a macromolecule. The QR codes at the end of each chapter give you access to the videos in French, that are part of the MOOC. English subtitles are available for all the videos. In the early stages of learning biological crystallography, the digital version of this book can therefore be a particularly useful companion.

 - Marie-Hélène Le Du, Pierre Legrand, Serena Sirigu, Sylvain Ravy - EDP Sciences & Science Press Introduction to biological crystallography

link: https://bit.ly/3zBC6p5

Early Steps of DNA Recombination: AlphaFold2 breaks a lock.

The human MRN nuclease, formed by the association of two proteins, Mre11 and Rad50, is a key player in homologous recombination and meiosis, two processes that utilize DNA break repair mechanisms. In eukaryotes, the Mre11 nuclease is equipped with a molecular lock that controls the activation of the enzyme: its activation is triggered during certain phases of the cell cycle (entry into G2, when DNA is duplicated to allow for the recombination process), when the lock is phosphorylated. This locking protein is called CtIP in humans and Sae2 in the yeast Saccharomyces cerevisiae. It is a protein with a largely disordered structure, which remained a significant challenge for structural characterization for a number of years.

In a study published in Molecular Cell, teams from I2BC, the Curie Institute, and IRB shed light on how the human MRN nuclease functions. The I2BC team modeled the structure of the yeast MRN complex using the AlphaFold2 algorithm. Interestingly, the program hesitates when modeling between two states: inactive (auto-inhibited) and active. Notably, the addition of phosphorylated Sae2 favors the active conformation in AlphaFold2’s predictions: Sae2 appears to unlock the MRN complex by establishing a synergistic set of interactions centered on the phosphorylation of a serine residue in Sae2. The structural predictions obtained with AlphaFold2, supported by in vitro and in vivo experiments, highlight that the phosphorylated Sae2/CtIP protein creates a network of interactions with MRN that promotes the release of its auto-inhibition.

All these findings illustrate how an apparently disordered protein can lift the auto-inhibition of a nuclease and thus control the switch between different repair pathways, which is important in humans for cell cycle progression and meiosis.

More information: https://www.sciencedirect.com/science/article/pii/S1097276524004428?via%3Dihub

Contact:  Raphaël GUEROIS  raphael.guerois@i2bc.paris-saclay.fr

Researchers from the AMIG team (I2BC department), in collaboration with the IRB (Switzerland), have modeled the interaction between HROB and the helicases MCM8-MCM9, some mutations of which predispose individuals to infertility or cancer. They demonstrate that HROB promotes the catalytic activity of the MCM8-MCM9 complex but does not play a role in its recruitment or stability.

The proteins MCM8 and MCM9 have recently been discovered as involved in multiple processes, normal and pathological, related to DNA replication, meiosis, homologous recombination, and mismatch repair. These proteins are helicases that have the ability to move along DNA and separate the two DNA strands. Variants of these proteins may predispose carriers to disorders such as infertility and cancer. In 2019, a third partner, HROB, was identified as associated with these two helicases without its mechanisms of action being understood. Since HROB is capable of interacting with DNA, three functional hypotheses can be considered:
1) HROB participates in the recruitment of helicases on DNA,
2) HROB stabilizes the assembly of MCM8 and MCM9 on DNA,
3) HROB promotes the catalytic activity of pre-assembled helicases.

In a study published in the journal Nature Communications, researchers from the AMIG team at the Institute of Integrative Biology of the Cell (I2BC) collaborated with a team from the Institute of Research in Biomedicine (IRB) in Switzerland to establish different structural models of the MCM8-MCM9-HROB complex. Guided by these models, a set of simple and compensatory mutations allowed the dissection of the functional role of HROB. The teams showed that only the third hypothesis was correct and that HROB is an essential factor for activating the MCM8-MCM9 helicase but not for its recruitment or stability. The structural model suggests that by transiently altering the conformation of a MCM9 subunit, HROB stimulates the translocation of the helicase along the DNA. Based on this model, it was even possible to design mutations that increase the efficiency of the helicase in the presence of HROB.

More information: https://www.nature.com/articles/s41467-024-47936-8

Contact: Raphaël Guerois raphael.guerois@i2bc.paris-saclay.fr 

Plants can benefit from fast nucleoside breakdown

Cells save their energy during nitrogen starvation by selective autophagy of ribosomes and degradation of RNA to ribonucleotides and nucleosides. Nucleosides are hydrolyzed by nucleoside N-ribohydrolases (nucleosidases, NRHs). Subclass I of NRHs preferentially hydrolyzes the purine ribosides while subclass II is more active towards uridine and xanthosine. Here, we performed a crystallographic and kinetic study to shed light on nucleoside preferences among plant NRHs followed by in vivo metabolomic and phenotyping analyses to reveal the consequences of enhanced nucleoside break-down. We report the crystal structure of Zea mays NRH2b (subclass II) and NRH3 (subclass I) in complexes with the substrate analog forodesine. Purine and pyrimidine catabolism are inseparable because nucleobase binding in the active site of ZmNRH is mediated via a water network and is thus unspecific. Dexamethasone-inducible ZmNRH overexpressor lines of Arabidopsis thaliana, as well as double nrh knockout lines of moss Physcomitrium patents, reveal a fine control of adenosine in contrast to other ribosides. ZmNRH overexpressor lines display an accelerated early vegetative phase including faster root and rosette growth upon nitrogen starvation or osmotic stress. Moreover, the lines enter the bolting and flowering phase much earlier. We observe changes in the pathways related to nitrogen-containing compounds such as β-alanine and several polyamines, which allow plants to reprogram their metabolism to escape stress. Taken together, crop plant breeding targetting enhanced NRH-mediated nitrogen recycling could therefore be a strategy to enhance plant growth tolerance and productivity under adverse growth conditions.

more information: https://onlinelibrary.wiley.com/doi/10.1111/tpj.16572

contact: Solange Morera solange.morera@i2bc.paris-saclay.fr

Quality trumps quantity: photoenzymatic decarboxylation of fatty acids does indeed occur in 300 ps. Despite the dismissal by our "slightly overproductive" competitors, citing it as 30x slower, our original findings stand strong!

In fatty acid photodecarboxylase (FAP), light-induced formation of the primary radical product RCOO● from fatty acid RCOO– occurs in 300 ps, upon which CO2 is released quasi-immediately. Based on the hypothesis that aliphatic RCOO●(spectroscopically uncharacterized because unstable) absorbs in the red similarly to aromatic carbonyloxy radicals such as 2,6-dichlorobenzoyloxy radical (DCB●), much longer-lived linear RCOO● has been suggested recently. We performed quantum chemical reaction pathway and spectral calculations. Thesecalculations are in line with the experimental DCB● decarboxylation dynamics and spectral properties and show that in contrast to DCB●, aliphatic RCOO● radicals a) decarboxylate with a very low energetic barrier and on the timescale of a few ps and b) exhibit little red absorption. A time-resolved infrared spectroscopy experiment confirms very rapid, <<300 ps RCOO● decarboxylation in FAP. We argue that this property is required for the observed high quantum yield of hydrocarbons formation by FAP.

more information: https://onlinelibrary.wiley.com/doi/10.1002/anie.202401376 

contact: Pavel MULLER <pavel.muller@i2bc.paris-saclay.fr>

The AMIG team at I2BC, in collaboration with teams from the Institut Curie and the Synchrotron Soleil, shows that the CAF-1 protein combines in its spatial organization flexible regions and rigid modules to deposit histones on DNA and effectively couple this process to DNA synthesis.

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.

More information: https://elifesciences.org/articles/91461

Contact: Françoise Ochsenbein francoise.ochsenbein@i2bc.paris-saclay.fr

The structural characterization of the catalytic cycle of the lipid transporter ATP8B1 by cryoEM reveals fundamental aspects of its substrate specificity and of its regulation.

Lipids are the main constituents of our cell membranes, which are formed as lipid bilayers. The distribution of lipids is far from uniform; it is asymmetric, with different lipid compositions in the inner and outer leaflet. This asymmetry is essential for a variety of cellular functions, from maintaining membrane homeostasis to enabling cell signaling and numerous other physiological processes including apoptosis and coagulation.

P4-ATPases, also known as flippases, are key players in creating and maintaining this lipid asymmetry. These enzymes actively transport lipids from the outside (exoplasmic) leaflet to the inside (cytosolic) leaflet coupled to ATP hydrolysis and ensure the proper distributions of lipids. The ATP8B1-CDC50A flippase complex in particular, has been the subject of the current study, is involved in a rare inherited liver disease called intrahepatic cholestasis. The function of ATP8B1 lipid flippase is critical for the regulation of bile production, a vital substance in our digestive system, but the direct link within bile producing liver cells remains unknown. Additionally, recent studies have spotlighted the relevance of genetic variants in the regulatory segment of the ATP8B1 gene as a strong genetic marker for Alzheimer’s resilience.

In the new study, the research team employed state-of-the-art cryo-electron microscopy techniques to capture nine different states associated with the lipid transport and determine structures at 2.4 to 3.1 Å overall resolution for these conformations. These structural insights, combined with functional and computational studies, reveal the inner workings of the human flippase ATP8B1-CDC50A complex and also resolves earlier discrepancies about the ATP8B1 transport substrates. Additionally, this work also provides key understanding about fine regulation by specific regulatory lipids known as phosphoinositides and by its autoinhibition mechanism.

This work was recently awarded ”article of the month” by the Société Française de Biochimie et Biologie Moléculaire (SFBBM) in December 2023.

More information: https://www.nature.com/articles/s41467-023-42828-9

Contact: Thibaud DIEUDONNE <thibaud.dieudonne@i2bc.paris-saclay.fr>

Structural studies by X-ray cristallography and CryoEM combined with cellular analyses show the stabilizing role of IP6 in assembly of DNA double-strand break repair in human.

IP6, inositol hexaphosphate or phytic acid, is a product of cellular metabolism that binds to numerous proteins whose functions it regulates. In an article published in the journal Nucleic Acids Research, scientists from I2BC (UMR 9198, CNRS/CEA/UPSaclay, Gif-sur-Yvette), from lPBS (CNRS/Université Paul Sabatier) and from Institut for Structural and Chemical Biology (University of Leicester, UK) reveal how IP6 stabilizes the assembly of a complex responsible for DNA break repair in humans.

Many cancer therapies induce DNA double-strand breaks. A DNA double-strand break represents the most serious type of DNA damage to a cell, since it amounts to splitting a chromosome in two. The resulting chromosome fragments can be lost or lead to translocations if they are not quickly rejoined. Unrepaired breaks most often lead to cell death, a property exploited to eradicate tumor cells during radiotherapy or certain chemotherapies. Yet DNA double-strand break repair pathways exist, and their performance in tumors determines the efficacy of these therapies. The dominant system for repairing DNA double-strand breaks in human cells is Non-Homologous End Joining, or NHEJ, initiated by the Ku protein, which rapidly encircles the ends of the break and acts as a hub for the other proteins needed to weld these ends together.

Using two techniques for studying proteins at atomic scale (crystallography and cryo-electron microscopy), the scientists discovered how a small molecule produced by the body and sometimes used as a dietary supplement, inositol-hexaphosphate (IP6) or phytic acid, binds to the Ku protein.

The scientists then analyzed the effect of IP6 on break repair in human cells. They modified the Ku protein to block its binding to IP6, and thus understand how the latter acts. The Ku mutants revealed that the presence of IP6 on the Ku protein stimulates its binding to the XLF protein, an essential step in efficiently welding the DNA break (see the model below). IP6 thus plays a key role in stabilizing the large complex of NHEJ proteins required for DNA break repair. This fundamental work resolves the question raised two decades ago regarding the role of IP6 in DNA double-strand break repair. It also opens up therapeutic prospects by identifying a new region on the Ku protein that could be targeted by small molecules to block DNA break repair in tumor cells.

More information: https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkad863/7327077?login=false

Contact: Jean-Baptiste CHARBONNIER <jb.charbonnier@i2bc.paris-saclay.fr>

We reveal unprecedented mechanistic structural details of how the active site of bacterial Nucleotidyl Cyclase toxins is sequentially remodeled by cofactor and substrate binding and how they can accommodate both purine or pyrimidine nucleotides as substrate.

Nucleotidyl cyclase (NC) enzymes are important ubiquitous enzymes that catalyze the production of cyclic nucleotides, crucial second messengers involved in numerous signaling pathways. ExoY-like toxins are bacterial virulence factors produced by Gram-negative γ- and β-proteobacteria. They belong to a broader family of bacterial NC toxins. When delivered into eukaryotic cells, NC toxins alter host cell signalling by overproducing both cyclic purine (cAMP, cGMP) and pyrimidine (cCMP, cUMP) nucleotides. To become potent NC enzymes inside host cells, they bind to specific host cofactors. The molecular and mechanistic details underlying the activation and catalytic specificities of NC toxins are only partially understood. ExoY NC toxins utilize either polymerized or monomeric (G-actin) actin as cofactor.
Here, we (a team at I2BC collaborating with scientists at ICSN, CNRS, UPS, Gif-Sur-Yvette, and at Institute Pasteur, Paris) investigated ExoY-like NCs that are selective for G-actin. Our in vitro investigations first revealed the physiological cofactor at work within host eukaryotic cells. These ExoYs activate by capturing and sequestering the cytoskeletal G-actin-profilin complex, disrupting its regulatory functions in the dynamic assembly of actin (Fig. 1, steps B-C).
Using X-ray crystallography at SOLEIL synchrotron, we have intercepted several structural snapshots along the ExoY activation pathway by G-actin-profilin. These structural data reveal unprecedented mechanistic details of how the active site of all NC toxins undergoes progressive remodeling upon cofactor and substrate binding (Fig. 1, step C). They reveal how NC toxins can accommodate purine or pyrimidine nucleotides as substrates and clarify crucial aspects of their catalytic purinyl and pyrimidinyl cyclase reaction. These structural insights into the multi-step activation of NC toxins may open new avenues to inhibit specifically this class of toxic bacterial NCs.

More information: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1011654

Contact: Louis RENAULT <louis.renault@i2bc.paris-saclay.fr>

This conference is postponed to a later date.

The next Cyclope conference organized by the CEA will be presented by Marie-Hélène Le DU at the INSTN at 8 p.m on the 24th of October 2023. The headline: “Proteins are everywhere".

They are found in animals as well as plants, bacteria or viruses. They allow the regular beating of your heart, the digestion of your last meal, maintain your body at a constant temperature, participate in the fight against infections that threaten you and much more...
Proteins are everywhere life exists and science has studied them for decades, but they are so numerous and so diverse that their world has yet to be explored. We invite you to discover this exciting research in the company of Marie-Hélène Le Du, CEA researcher at the Institute of Integrative Cell Biology (DRF/Joliot/I2BC), with the participation of Rémi Ruedas, a doctoral student at I2BC.

This study shows that DciA, the bacterial replicative helicase loader, promotes condensates in the presence of DNA. This opens the way to the possibility of non-membrane compartments in bacteria. The goal could be to concentrate the players involved in replication and thus facilitate it.

The loading of the bacterial replicative helicase DnaB is an essential step for genome replication and depends on the assistance of accessory proteins. Several of these proteins have been identified across the bacterial phyla. DciA is the most common loading protein in bacteria, yet the one whose mechanism is the least understood. We have previously shown that DciA from Vibrio cholerae is composed of a globular domain followed by an unfolded extension and demonstrated its strong affinity for DNA. Here, drawing on the skills of two I2BC facilities, Light microscopy (Imagerie-Gif) and PIM (Structural biology), we characterize the condensates formed by VcDciA upon interaction with a short single-stranded DNA substrate. We demonstrate the fluidity of these condensates using light microscopy and address their network organization through electron microscopy, thereby bridging events to conclude on a liquid-liquid phase separation behavior. Additionally, we observe the recruitment of DnaB in the droplets, concomitant with the release of DciA. We show that the well-known helicase loader DnaC from Escherichia coli is also competent to form these phase-separated condensates in the presence of ssDNA. Our phenomenological data are still preliminary as regards the existence of these condensates in vivo, but open the way for exploring the potential involvement of DciA in the formation of non-membrane compartments within the bacterium to facilitate the assembly of replication players on chromosomal DNA.

More information : https://pubmed.ncbi.nlm.nih.gov/39603490/

Contact : Sophie CHERUEL sophie.quevillon-cheruel@i2bc.paris-saclay.fr  and Stéphanie MARSIN stephanie.marsin@i2bc.paris-saclay.fr

Ultrafast formation of triplet statates by singlet fission in water solution.

We provide evidence that singlet fission—a process where one excited state splits into two—can occur in water-soluble compounds. Specifically, we studied perylene-3,4,9,10-tetracarboxylate, a molecule that forms transient disordered dimers. Using a combination of advanced techniques like time-resolved absorption and fluorescence spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and theoretical modeling, we were able to detect the key features of singlet fission and map out the behavior of the molecular assemblies. Our results show that the constant structural changes within these dimers play a crucial role in steering the process toward either singlet fission or charge separation. The quantum efficiency of producing triplet states is over 100%, with these states lasting for nanoseconds—highlighting how disordered systems can drive highly efficient photophysical processes.

More information: https://pubs.rsc.org/en/content/articlehtml/2024/sc/d4sc04732j

Contact: Manuel LLANSOLA <manuel.llansola@I2bc.paris-saclay.fr>

A system of redox enzymes facilitates a series of reactions that regulate oxygen reduction at photosystem I in plants.

In a study published in Plant Physiology, scientists from I2BC and IPS2 studied the redox regulation of superoxide production at the photosynthetic electron transfer level (pseudocyclic flow). Plants must optimise their molecular strategies to adapt to the light conditions of their environment. Reactive oxygen species (ROS) can play a role as signalling molecules but can also be potentially harmful. To manage light-induced ROS and maintain proper photosynthetic function, thiol-dependent redox enzymes play a crucial role in the redox regulation of the chloroplast. It has been shown that the main redox enzymes involved in controlling the ROS generated during photosynthesis are m-type thioredoxins, NADPH-dependent reductase C (NTRC) and peroxiredoxin 2-Cys. The interaction of these enzymes functions as a redox regulatory network, where 2-Cys PRX can be reduced by NTRC and Trx but can also re-oxidise the reduced thioredoxins, thus providing a system for rapid acclimatisation to changes in the light regime. The researchers showed that the membrane localisation of Trx m and NTRC varies as a function of photoperiod. In addition, their results show that it is the PSI itself that is redox regulated. The new results of this study make it possible to generate a new model of PSI redox regulation. This model may guide further research into the redox regulation of alternative electron transport pathways under conditions of fluctuating light or abiotic stress.

More information: DOI: 10.1093/plphys/kiae501 

Contact: Anja KRIEGER-LISZKAY anja.liszkay@i2bc.paris-saclay.fr

Mark your calendars for Jun. 26-28, 2024! Join us at Ecole Polytechnique in Palaiseau for the 7th International Conference on Physics and Biological Systems, where top-notch scientists converge to explore the dynamic interplay between physics and life sciences.

The 7th International Conference on Physics and Biological Systems will be held on Jun. 26-28 2024 at Ecole Polytechnique in Palaiseau, in the south of Paris. It aims to bring together a broad range of physical and life scientists working at the interface between the two disciplines around in-depth talks by first-rate international speakers. Attendance will be limited to 200 participants. We look forward to welcoming you in Palaiseau!

more information: https://www.lptms.universite-paris-saclay.fr/physbio2024/

contact: Pavel MULLER <pavel.muller@i2bc.paris-saclay.fr>

A Glacios2 200 kV has been delivered to I2BC, heralding, together with a Titan Krios 300 kV at synchrotron SOLEIL, a new era for cryo-electron microscopy in the Paris-Saclay area

Cryo-electron microscopy (cryo-EM) has become an indispensable tool in structural biology, enabling researchers to unravel the complexities of biological macromolecules and understand their roles in various cellular processes and diseases. These last ten years the field has undergone a revolution, with technical advances that have made atomic resolution reconstructions of large macromolecular complexes possible (the so-called 'resolution revolution'), I2BC received the latest generation 200kV microscope, Glacios2, on March 27, 2024. Joint operation is planned with the 300kV Titan Krios also just delivered at synchrotron SOLEIL. This will allow researchers to study structures of living machines at work in fine detail, both for purified and/or reconstituted complexes and in cells. These two cryo-electron microscopes are gathering scientists from all over the Paris-Saclay area, sparking further interest in future developments at the interface beween structural and cellular biology.

contact: Stéphane Bressanelli (plt-cryoem@i2bc.paris-saclay.fr)

AlphaFold2 is revolutionising protein structure prediction and structural biology practices. However, it may prove less effective for certain protein assemblies, particularly when they depend on intrinsically disordered regions. In an article published in Nature Communications, researchers from the I2BC show that applying a fragmentation strategy to the protein partners of such assemblies very significantly improves AlphaFold2’s prediction capacity.

Mapping protein-protein interaction networks is essential for understanding the dynamics of cellular functions and their cross-regulation. Precise knowledge of interaction sites makes it possible to specifically perturb the proteins in these networks and understand the synergies and competitions that ensure cell function.

Unfortunately, a great amount of structural information is still lacking to provide a detailed understanding of the organisation of interaction networks. The AlphaFold2 artificial intelligence programme has demonstrated a remarkable ability to predict the structures of protein assemblies that have co-evolved over long time scales. Its performance remained poorly characterised for assemblies involving intrinsically disordered regions, which often mediate transient and dynamic interactions during evolution.

In a study published in the journal Nature Communications, researchers from the AMIG team at the Institut de Biologie Intégrative de la Cellule – I2BC (CNRS/CEA/UPSaclay, Gif-sur-Yvette) have shown that AlphaFold2 performs poorly if large disordered regions are used directly for prediction (40% success rate). A protein fragmentation strategy was found to be particularly well adapted to predicting the interfaces between folded domains and small protein motifs that fold on contact with the partner. Applied on a large scale using the Jean Zay HPC infrastructure on more than 900 complexes, this strategy achieved a success rate of almost 90%, a very encouraging result for the systematic screening of protein interaction networks. Nevertheless, the study calls for vigilance with regard to the risks of detecting false positives, which will be at the heart of future developments in artificial intelligence strategies such as AlphaFold2.

More information: https://www.nature.com/articles/s41467-023-44288-7

Contact: Jessica ANDREANI and Raphaël GUEROIS  <jessica.andreani@i2bc.paris-saclay.fr> <raphael.guerois@i2bc.paris-saclay.fr>

Molecular movies? No problem for time-resolved serial femtosecond X-ray crystallography! We have obtained 18 snapshots visualizing the whole DNA repair by a photolyase: from the initial electron transfer to the active site recovery and DNA reannealing.

Photolyases, a ubiquitous class of flavoproteins, use blue light to repair DNA photolesions. In this work, we determined the structural mechanism of the photolyase-catalyzed repair of a cyclobutane pyrimidine dimer (CPD) lesion using time-resolved serial femtosecond crystallography (TR-SFX). We obtained 18 snapshots that show time-dependent changes in four reaction loci. We used these results to create a movie that depicts the repair of CPD lesions in the picosecond-to-nanosecond range, followed by the recovery of the enzymatic moieties involved in catalysis, completing the formation of the fully reduced enzyme-product complex at 500 nanoseconds. Finally, back-flip intermediates of the thymine bases to reanneal the DNA were captured at 25 to 200 microseconds. Our data cover the complete molecular mechanism of a photolyase and, importantly, its chemistry and enzymatic catalysis at work across a wide timescale and at atomic resolution.

More information: https://www.science.org/doi/10.1126/science.add7795

Contact: Pavel MULLER  <pavel.muller@i2bc.paris-saclay.fr>

A short disordered and conserved motif of the tumor suppressor BRCA2 triggers the assembly of a large ring-shaped 24-mer of the meiotic HSF2BP protein, whereas the disordered C-terminal motif of the meiotic BRME1 dissociates this ring complex.

In meiotic homologous recombination (HR), BRCA2 facilitates loading of the recombinases RAD51 and DMC1 at the sites of double-strand breaks (DSB). The HSF2BP-BRME1 complex interacts with BRCA2. Its absence causes a severe reduction in recombinase loading at meiotic DSB. We previously showed that, in somatic cancer cells ectopically producing HSF2BP, DNA damage can trigger HSF2BP-dependent degradation of BRCA2, which prevents HR. Here we report that, upon binding to BRCA2, HSF2BP forms octameric rings that are able to assemble into a large ring-shaped 24-mer. Addition of BRME1 leads to dissociation of both of these ring structures, 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 HR.

More information: https://www.science.org/doi/10.1126/sciadv.adi7352

Contact: Sophie ZINN JUSTIN <sophie.zinn@i2bc.paris-saclay.fr>

Identification of a novel mechanism for the regulation of the stathmin:tubulin interaction.

Microtubule dynamics is modulated by many cellular factors including stathmin family proteins. Vertebrate stathmins sequester two ab-tubulin heterodimers into a tight complex that cannot be incorporated in microtubules. Stathmins are regulated at the expression level during development and among tissues; they are also regulated by phosphorylation. We have studied the dissociation kinetics of tubulin:stathmin assemblies in presence of different tubulin-binding proteins and identified a critical role of the C-terminus of the stathmin partner. Destabilizing this C-terminal region may represent an additional regulatory mechanism of the interaction with tubulin of stathmin proteins.

More information: https://doi.org/10.1016/j.bbrc.2023.10.023

Contact: Benoît GIGANT <benoit.gigant@i2bc.paris-saclay.fr>

Kinases are exquisite drug targets, but usually too large for any NMR characterization at decent concentrations; we combined a number of innovative strategies enabling S/N~10 in 1h using less than 100 ug of protein.

We aimed at designing and testing affordable strategies for improving NMR sensitivity on common-size drug targets, e.g. kinases of 40 kDa. This goes notably via novel isotope labeling schemes, which we sought to be as versatile as possible in terms of recombinant expression organism.
We evaluated the benefits of the deuteration on alpha- and beta-positions of amino acids for a typical middle size protein domain, namely the model 40 kDa-large kinase p38α.
We used cystathionine gamma-synthase (and new high-performance mutants thereof) to execute position-specific deuteration of free amino acids by H/D exchange. Then, we employed cell-free expression in bacterial extracts to avoid any scrambling and back-protonation of the tested isotopically labelled amino acids (Ala, Leu, Lys, Ser, Asp, Glu, Gly).
Altogether, these combined strategies yielded a ten-fold sensitivity increase, as compared to the former classical NMR approaches. It can be applied using recombinant expression in cell-free systems and in eukaryotic cells. This allows recording residue-resolved solution 1H-15N NMR spectra of 100 μg of purified p38α in one hour with S/N∼10, and a residue-specific characterization of drug binding.
We want to apply this approach for in-cell NMR studies in the future.

More information: https://www.sciencedirect.com/science/article/pii/S2666441023000341?via%3Dihub

Contact: Theillet Francois-Xavier <francois-xavier.theillet@i2bc.paris-saclay.fr>