Biology subjects

Bouvignies, G.

Publications and source records attributed to Bouvignies, G..

4 recordsLinked to original sources

bioRxiv2024

KIF2C-induced nuclear condensation concentrates PLK1 and phosphorylated BRCA2 at the kinetochore microtubules in mitosis

During mitosis, the human microtubule depolymerase KIF2C increases the turnover of kinetochore-microtubule attachments. This facilitates the correction of attachment errors. Moreover, BRCA2 phosphorylated at Thr207 by PLK1 (BRCA2-pT207) assembles a complex including PLK1, PP2A and BUBR1 that contributes to the stability of the kinetochore-microtubule attachments. PLK1, together with Aurora B, critically regulate the accurate segregation of chromosomes. Here we demonstrate that KIF2C contains an N-terminal domain that binds directly to several phosphorylated peptides, including BRCA2-pT207. Using an optogenetic platform, we reveal that KIF2C assembles into membrane-less compartments or biomolecular condensates that are located next to microtubules. We provide evidence that condensate assembly depends on the presence of the newly defined N-terminal phospho-binding domain of KIF2C and on the kinase activities of Aurora B and PLK1. Moreover, KIF2C condensates concentrate active PLK1 and colocalize with BRCA2-pT207. We propose that, because of its phospho-dependent binding and oligomerization capacities, KIF2C forms biomolecular condensates that partition PLK1 and locally amplify its kinase activity during mitosis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/589357v1_ufig1.gif" ALT="Figure 1"> View larger version (80K): org.highwire.dtl.DTLVardef@3ff5bforg.highwire.dtl.DTLVardef@11efbdcorg.highwire.dtl.DTLVardef@1295118org.highwire.dtl.DTLVardef@18b3dd3_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Multivalent interactions of the disordered regions of XLF and XRCC4 foster robust cellular NHEJ and drive the formation of ligation-boosting condensates in vitro

In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). During repair, the Ku70/80 heterodimer (Ku), XRCC4 in complex with DNA Ligase 4 (X4L4), and XLF form a flexible scaffold that holds the broken DNA ends together. Insights into the architectural organization of the NHEJ scaffold and its regulation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) have recently been obtained by single-particle cryo-electron microscopy analysis. However, several regions, especially the C-terminal regions (CTRs) of the XRCC4 and XLF scaffolding proteins, have largely remained unresolved in experimental structures, which hampers the understanding of their functions. Here, we used magnetic resonance techniques and biochemical assays to comprehensively characterize the interactions and dynamics of the XRCC4 and XLF CTRs at atomic resolution. We show that the CTRs of XRCC4 and XLF are intrinsically disordered and form a network of multivalent heterotypic and homotypic interactions that promotes robust cellular NHEJ activity. Importantly, we demonstrate that the multivalent interactions of these CTRs led to the formation of XLF and X4L4 condensates in vitro which can recruit relevant effectors and critically stimulate DNA end ligation. Our work highlights the role of disordered regions in the mechanism and dynamics of NHEJ and lays the groundwork for the investigation of NHEJ protein disorder and its associated condensates inside cells with implications in cancer biology, immunology and the development of genome editing strategies.

biophysics↗

bioRxiv2023

Multivalent dynamic colocalization of avian influenza polymerase and nucleoprotein by intrinsically disordered ANP32A reveals the molecular basis of human adaptation

Adaptation of avian influenza RNA polymerase (FluPol) to human cells requires mutations on the 627-NLS domains of the PB2 subunit. The E627K adaptive mutation compensates a 33-amino-acid deletion in the acidic intrinsically disordered domain of the host transcription regulator ANP32A, a deletion that restricts FluPol activity in mammalian cells. The function of ANP32A in the replication transcription complex and in particular its role in host restriction remain poorly understood. Here we characterise ternary complexes formed between ANP32A, FluPol and the viral nucleoprotein, NP, supporting the putative role of ANP32A in shuttling NP to the replicase complex. We demonstrate that while FluPol and NP can simultaneously bind distinct linear motifs on avian ANP32A, the deletion in the shorter human ANP32A blocks this mode of colocalization. NMR reveals that NP and human-adapted Pol, containing the E627K mutation, simultaneously bind the identical extended linear motif on human ANP32A, in an electrostatically driven, highly dynamic and multivalent ternary complex. This study reveals a probable molecular mechanism underlying host adaptation, whereby E627K, which enhances the basic surface of the 627 domain, is selected to confer the necessary multivalent properties to allow ANP32A to colocalize NP and FluPol in human cells.

biochemistry↗

bioRxiv2021

Global Dynamics as Communication Sensors in Peptide Synthetase Cyclization Domains.

Structural biology is the foundation for deriving molecular mechanisms, where snapshots of macromolecules and binding partners inform on mutations that test or modify function. However, frequently, the impact of mutations violates the underpinnings of structural models, and mechanisms become cryptic. This conundrum applies to multidomain enzymatic systems called nonribosomal peptide synthetases (NRPSs), which assemble simple substrates into complex metabolites often with pharmaceutical properties. Engineering NRPSs can generate new pharmaceuticals1-3 but a dynamic domain organization challenges rational design.4-8 Using nuclear magnetic resonance (NMR), we determined the solution structure of a 52 kDa cyclization domain and demonstrate that global intra-domain dynamics enable sensing of substrates tethered to partner domains and draw an allosteric response encompassing the enzymes buried active site and two binding sites 40 [A] apart. We show that a point-site mutation that impedes the domain dynamics globally hampers the allosteric response. We demonstrate this mechanism through NMR experiments that provide atomic-level read-outs of allosteric responses during biochemical transformations in situ. Our results establish global structural dynamics as sensors of molecular events that can remodel domain interactions and illustrate the need for integrating structural dynamics explicitly when deriving molecular mechanisms through mutagenesis and structural biology.

biophysics↗