Nse5/6 inhibits the Smc5/6 ATPase and modulates DNA substrate binding

Article30 June 2021Open Access Source DataTransparent process Nse5/6 inhibits the Smc5/6 ATPase and modulates DNA substrate binding Michael Taschner orcid.org/0000-0002-8881-3705 Department of Fundamental Microbiology (DMF), Faculty Biology Medicine (FBM), University Lausanne (UNIL), Lausanne, Switzerland Search for more papers by this author Jérôme Basquin Max Planck Institute Biochemistry, Martinsried, Germany These authors contributed equally to work Barbara Steigenberger Biomolecular Mass Spectrometry Proteomics, Bijvoet Center Research Utrecht Pharmaceutical Sciences, University, Utrecht, The Netherlands Proteomics Centre, Ingmar B Schäfer Young-Min Soh Claire Esben Lorentzen orcid.org/0000-0001-6493-7220 Molecular Genetics, Aarhus Aarhus, Denmark Markus Räschle Kaiserslautern, Richard A Scheltema Stephan Gruber Corresponding Author [email protected] orcid.org/0000-0002-0150-0395 Information Taschner1, Basquin2, Steigenberger2,3,4, Schäfer2, Soh1, Lorentzen5, Räschle6, Scheltema3,4 *,1 1Department 2Max 3Biomolecular 4Netherlands 5Department 6Molecular **Corresponding author. Tel: +41 21 692 5601; E-mail: EMBO Journal (2021)40:e107807https://doi.org/10.15252/embj.2021107807 PDFDownload PDF article text main figures. Peer ReviewDownload a summary editorial decision including letters, reviewer comments responses feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Eukaryotic cells employ three SMC (structural maintenance chromosomes) complexes control folding topology. complex plays roles in repair preventing accumulation deleterious junctions. To elucidate how specific features govern these functions, we reconstituted yeast holo-complex. We found that sub-complex strongly inhibited productive ATP binding. This inhibition was relieved plasmid but not short linear DNA, while opposing effects were observed without Nse5/6. uncovered two sites on Smc5/6, based an crystal structure cross-linking mass spectrometry data. One site is located at arms one heads, latter likely exerting inhibitory hydrolysis. Cysteine demonstrated interaction with anchored domains non-productive state, which destabilized DNA. Under similar conditions, Nse4/3/1 module detached from ATPase. Altogether, show selection modulated direct SYNOPSIS prevents toxic junctions during replication repair, order enable faithful chromosome segregation mitosis meiosis. Biochemical reconstitution holo-complex reveals central role selection. promotes ATP-dependent, salt-stable association Smc5/6. absence contacts hexamer via multiple interfaces joint heads. Lysine (XL-MS) cysteine uncover major conformational changes upon association. core shows HEAT-repeat-like organization. Introduction Maintenance transmission genetic information are essential processes all life orchestrated widely conserved structural chromosomes (SMC) [reviewed (Yatskevich et al, 2019)]. function ATP-powered DNA-folding machines bacteria prevent entanglement newly replicated ensure their unperturbed daughter cells. also need such entanglements cell division, additionally efficient sister chromatid cohesion, condensation individualization be ensured. Three distinct eukaryotic (cohesin, condensin Smc5/6) divide tasks between them. Cohesin folds interphase into defined regulate gene expression (Szabo 2019) participates homologous recombination (Litwin 2018). It holds chromatids together S-phase onset anaphase 2019). Condensin compacts structures promote resolution disjunction prometaphase anaphase, respectively (Hirano, 2016). molecular functions understood less detail (Aragon, Several genes first identified screens damage-sensitive mutants (Prakash Prakash, 1977; Lehmann 1995; McDonald 2003; Onoda 2004; Torres-Rosell 2005a; 2005b). Complete loss leads death associated severe defects both mitotic meiotic divisions (McDonald Pebernard Copsey 2013; Xaver 2013). Without certain as unresolved intermediates, intertwinings, or incompletely chromosomal regions proper (Torres-Rosell 2005b; 2007; Kegel 2011), especially repeated sequences ribosomal arrays (Peng however unclear whether formation removal. At have dimer proteins, each contains “hinge” domain mediates dimerization connects long (35–50 nm) antiparallel coiled-coil “arm” globular ABC-type “head” highly motifs hydrolysis (Hirano 2001; Lammens Hopfner, Two molecules sandwiched residues Walker subunit signature motif other subunit. heads complexes, rather slow (mostly < 1 ATP/s) compared ATPases. stimulated presence substrates (Hassler 2018)] (Fousteri Lehmann, 2000). cycle involves rearrangements within dimers, been delineated only some detail, example, prokaryotic Smc-ScpAB (Soh 2015; Diebold-Durand 2017; Burmann 2019; Chapard Vazquez Nunez Lee 2020). Briefly, proteins “juxtaposed” J-state closely aligned clear rod-shaped appearance electron micrographs (Diebold-Durand contact close form incompatible In ATP, head rearrange adopt “ATP-engaged” E-state (Lammens 2017). conformation rod conformation. least, it opens head-proximal area, yielding open ring-like has cohesin cryo-EM (Higashi 2020; Shi 2020) characterized paramagnetic resonance (Vazquez 2021). Upon hydrolysis, disengage, coiled coils zip back up, reverts J-state. presumably assists transitions, leading positive effect rate. Indeed, top engaged described several related (Liu 2016; Seifert Higashi Another invariably subunit, called “kleisin”, asymmetrically bridges create tripartite ring capable entrapping its lumen (Haering Palecek 2006; Gligoris 2014; Wilhelm 2015). Kleisin serves attachment point additional KITE (Kleisin-Interacting Tandem winged-helix Element) HAWK (HEAT-protein Associated With Kleisin) families (Palecek Gruber, Wells 2017) interactions regulation emerging (Zabrady Kschonsak Li 2018; Vondrova fully assembled utilizes energy released manners: (i) “Topological entrapment” inside SMC/kleisin after regulated opening (an) entry/exit gate(s) mechanism (Gligoris 2014). Similar activities (Cuylen 2011; Cuylen 2013), (Kanno Gutierrez-Escribano (Wilhelm 2015), albeit mechanistic detail. (ii) “Loop extrusion” refers active ATP-dependent used compact shape Such biochemical activity so far vitro (Ganji Davidson Kim may well feature pro- relatives. make (Smc5 Smc6) six “Non-SMC Elements” (Nse1-6) profoundly different kleisin Nse4 comparatively small protein. binds interactors (Nse1 Nse3) belonging family than Doyle 2010; Nse2 attaches arm Smc5. lacks known relatives (Duan 2009a). Apart Smc5 Smc6 harbours ubiquitin-related modifier (SUMO) ligase (Andrews 2005; Zhao Blobel, 2005) ubiquitin Nse1 (Doyle 2010). Mutants disruptions enzymatic viable sensitive damage, thus required non-essential Potts Yu, 2008). removal any subunits lethal 2003). last subunits, Nse5 Nse6, stable heterodimer very weak sequence similarity presumed vertebrate counterparts Slf1 Slf2 (Raschle Experiments performed budding fission yielded disparate results. While reported bind region S. pombe 2006), shown hinge cerevisiae 2009b). Recent mapping experiments human Slf1/2 showed mode (Adamus neither nor Nse6 (Pebernard they viability even under conditions (Zhao Aragon, 2018) (Fig 3D). involved Bustard 2012), potentially working SUMOylation (Bustard 2016)). recruiting damage through N-terminal unstructured peptide multi-BRCT Rtt107 (Leung Wan Single-molecule tracking recently suggested loading (Etheridge Nse5/6, determined investigated influence binding, inducing rearrangement domains. Addition molecules, robustly hexamer, suggesting organization holo-complex, (XL-MS). data revealed involving kleisin/KITE summary, our demonstrate key partner associates allow modulate rate response Results Reconstitution octamer Here, focused analysis “loader” context do so, separately origin corresponding co-expression E. coli. enriched affinity purification using Twin-Strep-tag Smc6, purified His-tag Nse5. protein preparations further polished ion exchange chromatography gel filtration. Both eluted analytical filtration column single species apparently stoichiometric composition 1A). Mixing prior resulted shift smaller elution volume, indicating octamer. Figure 1. architecture cryo-electron microscopy (cryo-EM) complexes. Left panel, schematic depiction “core” dimer. Middle profiles (Superose 6 Increase 3.2/300) obtained mixing hexamer. Measured absorption 280 nm. Right peak fractions analysed SDS–PAGE Coomassie Brilliant Blue staining. Circular representation lysine–lysine inter-subunit cross-links buffer containing 250 mM NaCl. For simplicity, (or domains) grouped, thickness lines indicates total number particular type. divided N- C-terminal (HD) (CC) domain. full individual intra-subunit cross-links, see Dataset EV1. Cross-links cross-link did match elongated particle displayed dashed line red colours. cartoon right folded examples intra- inter-links indicated white black lines, respectively. Selected 2D class averages (left images). Representative classes dimers hexamers (images 3 4). Emerging details schematically (right panel). available online figure. Data 1A [embj2021107807-sup-0003-SDataFig1A.tif] Download figure PowerPoint determine overall octamer, lysine-specific XL-MS identify proximity. Using short-spacer, enrichable, lysine-reactive reagent PhoX (Steigenberger 2019), detected 98 (intra-links) 64 (inter-links) NaCl 1B). Additional another (see below). pattern intra-links generally good agreement what expected 1C). sizeable fraction supporting notion co-alignment juxtaposed (J-state) (in colour) deviated pattern, could represent false hit cut off 1% formed proteins. largely comparable results discussed below. below, structure. investigate putative complementary method, cryogenic microscopy. particles length about 45 nm, consistent extended co-aligned 1D, images 2). An density along noticeable anticipated position Near domains, recognizable, often slightly bent disruption near Of note, included dimeric forms head-to-head configuration relevance remains determined. Crystal Structural currently limited disordered N-terminus interacts (Wan 2A). Compounding this, there conflicting reports literature ?-helical HEAT repeats relatedness Slf2, Slf1/Slf2 Raschle Adamus Yu Limited proteolysis 2B) led design N-terminally truncated fragments (154-C 177-C) easily co-purified Nse5/Nse6(177-C) selenomethionine substituted crystals diffracted 3.3 Å provided experimental phase compute high enough quality build model (Figs 2C EV1) (Table 1). map, 284–464 visible, predicted secondary indeed 11 ?-helices, arranged crescent-shaped EV1A). observe reliable 177–283, flexibility crystal. Nse5, 16 ?-helices 2–518 EV1B), helices being connected loops (147–197, 290–340 430–491), poor none all. 2. co-crystal Domain Putative boundaries prediction denoted arrowheads, colours when producing fragments, otherwise grey preparation Nse5/Nse6 trypsin. Samples taken time points indicated. Front view representation. elements orange purple colours, labelled. Conservation interface. Interaction surface panel) orientation, interface (middle Colour code residue conservation given bottom panel. area (bottom coding (D). loop lysine (K148) Fig 3). 2B [embj2021107807-sup-0004-SDataFig2B.tif] Click here expand views moiety front, side displays (at left concave interacting highest conservation. colour right. Display (A). Zoom helix ?12 preceding ?1 ?3. Verification BMOE cross-linking. Residues H368 G56 zoom-in left) mutated cysteines. Incubation (30 s) mutant wild-type robust seen decrease electrophoretic mobility cross-linked (right). Superimposition ?8-?11 selected hits DALI search Protein Bank front view. Root mean square displacement (rmsd) values indicator fit. (helices ?1- ?6). (this work; PDB: 7OGG) published progress (PDB: 7LTO). independently derived models (rmsd 0.829 Å). Table collection refinement statistics. Nse5/Nse6(177-C)* Wavelength 0.9792 Resolution range 19.85–3.293 (3.41–3.293) Space group P 2 Unit 99.116 147.368 74.444 90 Total reflections 91905 (7109) Unique 16988 (1639) Multiplicity 5.4 (4.3) Completeness (%) 99.09 (98.38) Mean I/sigma(I) 16.77 (1.98) Wilson B-factor 110.80 R-merge 0.07468 (0.6013) R-meas 0.08246 (0.6831) R-pim 0.03437 (0.318) CC1/2 0.999 (0.858) CC* (0.961) Reflections 16972 (1636) R-free 1700 (163) R-work 0.2971 (0.3900) 0.3075 (0.4063) CC(work) 0.885 (0.764) CC(free) 0.871 (0.728) Macromolecules 4934 658 RMS(bonds) 0.005 RMS(angles) 1.09 Ramachandran favoured 98.59 allowed 1.41 outliers 0.00 Statistics highest-resolution shell parentheses. covers around 1,300 Å2 2D), calculated PISA server (Krissinel Henrick, 2007). On mainly ?3, ?6, ?9 ?11 forming Its pronounced hydrophobic packs tightly against ?2 ?5, connecting ?4 ?5. Sidechains pocket engage polar contacts. Additional, conserved, exist ?3 EV1C). unmodelled towards might strengthen part Due large fact Nse