Publications by authors named "Sebastiano Pasqualato"

26 Publications

  • Page 1 of 1

Seroprevalence of SARS-CoV2 in IBD patients treated with biological therapy.

J Crohns Colitis 2020 Nov 19. Epub 2020 Nov 19.

Gastroenterology and Endoscopy Unit, Fondazione IRCCS Cà Granda, Ospedale Maggiore Policlinico, Milan, Italy.

Background And Aims: A similar course of COVID-19 in patients with inflammatory bowel diseases (IBD) and in the general population has been reported. However, disease prevalence in IBD patients is presently unknown. In this prospective observational study we aimed at determining SARS-CoV2 infection prevalence in IBD patients treated with biological therapy.

Methods: 354 sera from IBD patients under biological therapy recruited from three different locations in Italy and Germany were evaluated for antibody presence by RBD ELISA. Control groups were i) age-matched healthy subjects tested in the same time period in Milan, Italy; ii) healthy subjects collected in the pre-COVID era; iii) IBD patients under biological therapy collected in the pre-COVID era.

Results: 8 out of 354 patients tested positive for the anti-RBD-SARS-CoV2 IgG antibody (prevalence 2.3%). IgG positive patients' percentage recruited from Milan was significantly higher than those recruited from other locations (prevalence 5.4% vs. 0.4% p < 0.005). IgG positive patients reported a significantly higher incidence of fever, anosmia and ageusia, and were more likely to have entered in close contact with COVID-19 positive subjects before the study enrolment.

Conclusions: Seroprevalence of SARS-CoV2 in IBD patients treated with biological therapy reflects values measured in the local general population. Specific symptoms and contact history with SARS-CoV2-infected individuals strongly increase the likelihood of SARS-CoV2 seropositivity.
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http://dx.doi.org/10.1093/ecco-jcc/jjaa237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7717179PMC
November 2020

Structural Basis of Inhibition of the Pioneer Transcription Factor NF-Y by Suramin.

Cells 2020 10 29;9(11). Epub 2020 Oct 29.

Department of Biosciences, University of Milano, Via Celoria 26, 20133 Milano, Italy.

NF-Y is a transcription factor (TF) comprising three subunits (NF-YA, NF-YB, NF-YC) that binds with high specificity to the CCAAT sequence, a widespread regulatory element in gene promoters of prosurvival, cell-cycle-promoting, and metabolic genes. Tumor cells undergo "metabolic rewiring" through overexpression of genes involved in such pathways, many of which are under NF-Y control. In addition, NF-YA appears to be overexpressed in many tumor types. Thus, limiting NF-Y activity may represent a desirable anti-cancer strategy, which is an ongoing field of research. With virtual-screening docking simulations on a library of pharmacologically active compounds, we identified suramin as a potential NF-Y inhibitor. We focused on suramin given its high water-solubility that is an important factor for in vitro testing, since NF-Y is sensitive to DMSO. By electrophoretic mobility shift assays (EMSA), isothermal titration calorimetry (ITC), STD NMR, X-ray crystallography, and molecular dynamics (MD) simulations, we showed that suramin binds to the histone fold domains (HFDs) of NF-Y, preventing DNA-binding. Our analyses, provide atomic-level detail on the interaction between suramin and NF-Y and reveal a region of the protein, nearby the suramin-binding site and poorly conserved in other HFD-containing TFs, that may represent a promising starting point for rational design of more specific and potent inhibitors with potential therapeutic applications.
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http://dx.doi.org/10.3390/cells9112370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692634PMC
October 2020

Persistence of Anti-SARS-CoV-2 Antibodies in Non-Hospitalized COVID-19 Convalescent Health Care Workers.

J Clin Med 2020 Oct 1;9(10). Epub 2020 Oct 1.

Department of Experimental Oncology, European Institute of Oncology IRCCS, via Adamello 16, 20139 Milan, Italy.

Although antibody response to SARS-CoV-2 can be detected early during the infection, several outstanding questions remain to be addressed regarding the magnitude and persistence of antibody titer against different viral proteins and their correlation with the strength of the immune response. An ELISA assay has been developed by expressing and purifying the recombinant SARS-CoV-2 Spike Receptor Binding Domain (RBD), Soluble Ectodomain (Spike), and full length Nucleocapsid protein (N). Sera from healthcare workers affected by non-severe COVID-19 were longitudinally collected over four weeks, and compared to sera from patients hospitalized in Intensive Care Units (ICU) and SARS-CoV-2-negative subjects for the presence of IgM, IgG and IgA antibodies as well as soluble pro-inflammatory mediators in the sera. Non-hospitalized subjects showed lower antibody titers and blood pro-inflammatory cytokine profiles as compared to patients in Intensive Care Units (ICU), irrespective of the antibodies tested. Noteworthy, in non-severe COVID-19 infections, antibody titers against RBD and Spike, but not against the N protein, as well as pro-inflammatory cytokines decreased within a month after viral clearance. Thus, rapid decline in antibody titers and in pro-inflammatory cytokines may be a common feature of non-severe SARS-CoV-2 infection, suggesting that antibody-mediated protection against re-infection with SARS-CoV-2 is of short duration. These results suggest caution in using serological testing to estimate the prevalence of SARS-CoV-2 infection in the general population.
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http://dx.doi.org/10.3390/jcm9103188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7600936PMC
October 2020

Discovery of Reversible Inhibitors of KDM1A Efficacious in Acute Myeloid Leukemia Models.

ACS Med Chem Lett 2020 May 13;11(5):754-759. Epub 2020 Feb 13.

Department of Experimental Oncology, Academic Drug Discovery, European Institute of Oncology IRCCS, Via Adamello 16, 20139 Milan, Italy.

Lysine-specific demethylase 1 (LSD1 or KDM1A) is a FAD-dependent enzyme that acts as a transcription corepressor or coactivator by regulating the methylation status of histone H3 lysines K4 and K9, respectively. KDM1A represents an attractive target for cancer therapy. While, in the past, the main medicinal chemistry strategy toward KDM1A inhibition was based on the optimization of ligands that irreversibly bind the FAD cofactor within the enzyme catalytic site, we and others have also identified reversible inhibitors. Herein we reported the discovery of 5-imidazolylthieno[3,2-]pyrroles, a new series of KDM1A inhibitors endowed with picomolar inhibitory potency, active in cells and efficacious after oral administration in murine leukemia models.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236255PMC
May 2020

Organizational Principles of the NuMA-Dynein Interaction Interface and Implications for Mitotic Spindle Functions.

Structure 2020 07 14;28(7):820-829.e6. Epub 2020 May 14.

IEO, European Institute of Oncology IRCCS, 20141 Milan, Italy. Electronic address:

Mitotic progression is orchestrated by the microtubule-based motor dynein, which sustains all mitotic spindle functions. During cell division, cytoplasmic dynein acts with the high-molecular-weight complex dynactin and nuclear mitotic apparatus (NuMA) to organize and position the spindle. Here, we analyze the interaction interface between NuMA and the light intermediate chain (LIC) of eukaryotic dynein. Structural studies show that NuMA contains a hook domain contacting directly LIC1 and LIC2 chains through a conserved hydrophobic patch shared among other Hook adaptors. In addition, we identify a LIC-binding motif within the coiled-coil region of NuMA that is homologous to CC1-boxes. Analysis of mitotic cells revealed that both LIC-binding sites of NuMA are essential for correct spindle placement and cell division. Collectively, our evidence depicts NuMA as the dynein-activating adaptor acting in the mitotic processes of spindle organization and positioning.
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http://dx.doi.org/10.1016/j.str.2020.04.017DOI Listing
July 2020

Hexameric NuMA:LGN structures promote multivalent interactions required for planar epithelial divisions.

Nat Commun 2019 05 17;10(1):2208. Epub 2019 May 17.

IEO, European Institute of Oncology IRCCS, 20141, MILANO, Italy.

Cortical force generators connect epithelial polarity sites with astral microtubules, allowing dynein movement to orient the mitotic spindle as astral microtubules depolymerize. Complexes of the LGN and NuMA proteins, fundamental components of force generators, are recruited to the cortex by Gαi-subunits of heterotrimeric G-proteins. They associate with dynein/dynactin and activate the motor activity pulling on astral microtubules. The architecture of cortical force generators is unknown. Here we report the crystal structure of NuMA:LGN hetero-hexamers, and unveil their role in promoting the assembly of active cortical dynein/dynactin motors that are required in orchestrating oriented divisions in polarized cells. Our work elucidates the basis for the structural organization of essential spindle orientation motors.
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http://dx.doi.org/10.1038/s41467-019-09999-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6525239PMC
May 2019

Thieno[3,2-b]pyrrole-5-carboxamides as New Reversible Inhibitors of Histone Lysine Demethylase KDM1A/LSD1. Part 2: Structure-Based Drug Design and Structure-Activity Relationship.

J Med Chem 2017 03 27;60(5):1693-1715. Epub 2017 Feb 27.

Department of Experimental Oncology, Academic Drug Discovery, European Institute of Oncology , Via Adamello 16, 20139 Milano, Italy.

The balance of methylation levels at histone H3 lysine 4 (H3K4) is regulated by KDM1A (LSD1). KDM1A is overexpressed in several tumor types, thus representing an emerging target for the development of novel cancer therapeutics. We have previously described ( Part 1, DOI 10.1021.acs.jmedchem.6b01018 ) the identification of thieno[3,2-b]pyrrole-5-carboxamides as novel reversible inhibitors of KDM1A, whose preliminary exploration resulted in compound 2 with biochemical IC = 160 nM. We now report the structure-guided optimization of this chemical series based on multiple ligand/KDM1A-CoRest cocrystal structures, which led to several extremely potent inhibitors. In particular, compounds 46, 49, and 50 showed single-digit nanomolar IC values for in vitro inhibition of KDM1A, with high selectivity in secondary assays. In THP-1 cells, these compounds transcriptionally affected the expression of genes regulated by KDM1A such as CD14, CD11b, and CD86. Moreover, 49 and 50 showed a remarkable anticlonogenic cell growth effect on MLL-AF9 human leukemia cells.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01019DOI Listing
March 2017

Thieno[3,2-b]pyrrole-5-carboxamides as New Reversible Inhibitors of Histone Lysine Demethylase KDM1A/LSD1. Part 1: High-Throughput Screening and Preliminary Exploration.

J Med Chem 2017 03 27;60(5):1673-1692. Epub 2017 Feb 27.

Department of Experimental Oncology, Academic Drug Discovery, European Institute of Oncology , Via Adamello 16, 20139 Milano, Italy.

Lysine specific demethylase 1 KDM1A (LSD1) regulates histone methylation and it is increasingly recognized as a potential therapeutic target in oncology. We report on a high-throughput screening campaign performed on KDM1A/CoREST, using a time-resolved fluorescence resonance energy transfer (TR-FRET) technology, to identify reversible inhibitors. The screening led to 115 hits for which we determined biochemical IC, thus identifying four chemical series. After data analysis, we have prioritized the chemical series of N-phenyl-4H-thieno[3, 2-b]pyrrole-5-carboxamide for which we obtained X-ray structures of the most potent hit (compound 19, IC = 2.9 μM) in complex with the enzyme. Initial expansion of this chemical class, both modifying core structure and decorating benzamide moiety, was directed toward the definition of the moieties responsible for the interaction with the enzyme. Preliminary optimization led to compound 90, which inhibited the enzyme with a submicromolar IC (0.162 μM), capable of inhibiting the target in cells.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01018DOI Listing
March 2017

Purification and characterization of a DNA-binding recombinant PREP1:PBX1 complex.

PLoS One 2015 9;10(4):e0125789. Epub 2015 Apr 9.

FIRC (Foundation for Italian Cancer Research) Institute of Molecular Oncology (IFOM), via Adamello 16, 20139, Milan, Italy.

Human PREP1 and PBX1 are homeodomain transcriptional factors, whose biochemical and structural characterization has not yet been fully described. Expression of full-length recombinant PREP1 (47.6 kDa) and PBX1 (46.6 kDa) in E. coli is difficult because of poor yield, high instability and insufficient purity, in particular for structural studies. We cloned the cDNA of both proteins into a dicistronic vector containing an N-terminal glutathione S-transferase (GST) tag and co-expressed and co-purified a stable PBX1:PREP1 complex. For structural studies, we produced two C-terminally truncated complexes that retain their ability to bind DNA and are more stable than the full-length proteins through various purification steps. Here we report the production of large amounts of soluble and pure recombinant human PBX1:PREP1 complex in an active form capable of binding DNA.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0125789PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4391845PMC
April 2016

Fast native-SAD phasing for routine macromolecular structure determination.

Nat Methods 2015 Feb 15;12(2):131-3. Epub 2014 Dec 15.

Swiss Light Source at Paul Scherrer Institut, Villigen, Switzerland.

We describe a data collection method that uses a single crystal to solve X-ray structures by native SAD (single-wavelength anomalous diffraction). We solved the structures of 11 real-life examples, including a human membrane protein, a protein-DNA complex and a 266-kDa multiprotein-ligand complex, using this method. The data collection strategy is suitable for routine structure determination and can be implemented at most macromolecular crystallography synchrotron beamlines.
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http://dx.doi.org/10.1038/nmeth.3211DOI Listing
February 2015

Structural and functional framework for the autoinhibition of Nedd4-family ubiquitin ligases.

Structure 2014 Nov 30;22(11):1639-49. Epub 2014 Oct 30.

Max Planck Institute for Developmental Biology, Spemannstrasse 35, 72076 Tübingen, Germany. Electronic address:

Nedd4-family ubiquitin ligases are key regulators of cell surface receptor signaling. Their dysregulation is associated with several human diseases, including cancer. Under normal conditions, the activity of various Nedd4 E3s is controlled through an autoinhibitory interaction of the N-terminal C2 domain with the C-terminal catalytic HECT domain. Here, we report the structural and functional framework for this intramolecular interaction. Our nuclear magnetic resonance (NMR) data and biochemical analyses on Smurf2 and Nedd4 show that the C2 domain has the potential to regulate E3 activity by maintaining the HECT domain in a low-activity state where its ability for transthiolation and noncovalent Ub binding are impaired.
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http://dx.doi.org/10.1016/j.str.2014.09.006DOI Listing
November 2014

The pseudo GTPase CENP-M drives human kinetochore assembly.

Elife 2014 Jul 8;3:e02978. Epub 2014 Jul 8.

Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany Centre for Medical Biotechnology, Faculty of Biology, University of Duisburg-Essen, Essen, Germany

Kinetochores, multi-subunit complexes that assemble at the interface with centromeres, bind spindle microtubules to ensure faithful delivery of chromosomes during cell division. The configuration and function of the kinetochore-centromere interface is poorly understood. We report that a protein at this interface, CENP-M, is structurally and evolutionarily related to small GTPases but is incapable of GTP-binding and conformational switching. We show that CENP-M is crucially required for the assembly and stability of a tetramer also comprising CENP-I, CENP-H, and CENP-K, the HIKM complex, which we extensively characterize through a combination of structural, biochemical, and cell biological approaches. A point mutant affecting the CENP-M/CENP-I interaction hampers kinetochore assembly and chromosome alignment and prevents kinetochore recruitment of the CENP-T/W complex, questioning a role of CENP-T/W as founder of an independent axis of kinetochore assembly. Our studies identify a single pathway having CENP-C as founder, and CENP-H/I/K/M and CENP-T/W as CENP-C-dependent followers.DOI: http://dx.doi.org/10.7554/eLife.02978.001.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4080450PMC
http://dx.doi.org/10.7554/eLife.02978DOI Listing
July 2014

Modular assembly of RWD domains on the Mis12 complex underlies outer kinetochore organization.

Mol Cell 2014 Feb 13;53(4):591-605. Epub 2014 Feb 13.

Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany; Centre for Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Universitätsstrasse, 45141 Essen, Germany. Electronic address:

Faithful chromosome segregation is mandatory for cell and organismal viability. Kinetochores, large protein assemblies embedded in centromeric chromatin, establish a mechanical link between chromosomes and spindle microtubules. The KMN network, a conserved 10-subunit kinetochore complex, harbors the microtubule-binding interface. RWD domains in the KMN subunits Spc24 and Spc25 mediate kinetochore targeting of the microtubule-binding subunits by interacting with the Mis12 complex, a KMN subcomplex that tethers directly onto the underlying chromatin layer. Here, we show that Knl1, a KMN subunit involved in mitotic checkpoint signaling, also contains RWD domains that bind the Mis12 complex and that mediate kinetochore targeting of Knl1. By reporting the first 3D electron microscopy structure of the KMN network, we provide a comprehensive framework to interpret how interactions of RWD-containing proteins with the Mis12 complex shape KMN network topology. Our observations unveil a regular pattern in the construction of the outer kinetochore.
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http://dx.doi.org/10.1016/j.molcel.2014.01.019DOI Listing
February 2014

Exome sequence reveals mutations in CoA synthase as a cause of neurodegeneration with brain iron accumulation.

Am J Hum Genet 2014 Jan 19;94(1):11-22. Epub 2013 Dec 19.

Unit of Molecular Neurogenetics - Pierfranco and Luisa Mariani Center for the study of Mitochondrial Disorders in Children, IRCCS Foundation Neurological Institute "C. Besta," 20126 Milan, Italy. Electronic address:

Neurodegeneration with brain iron accumulation (NBIA) comprises a clinically and genetically heterogeneous group of disorders with progressive extrapyramidal signs and neurological deterioration, characterized by iron accumulation in the basal ganglia. Exome sequencing revealed the presence of recessive missense mutations in COASY, encoding coenzyme A (CoA) synthase in one NBIA-affected subject. A second unrelated individual carrying mutations in COASY was identified by Sanger sequence analysis. CoA synthase is a bifunctional enzyme catalyzing the final steps of CoA biosynthesis by coupling phosphopantetheine with ATP to form dephospho-CoA and its subsequent phosphorylation to generate CoA. We demonstrate alterations in RNA and protein expression levels of CoA synthase, as well as CoA amount, in fibroblasts derived from the two clinical cases and in yeast. This is the second inborn error of coenzyme A biosynthesis to be implicated in NBIA.
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http://dx.doi.org/10.1016/j.ajhg.2013.11.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3882905PMC
January 2014

Structure of a ubiquitin-loaded HECT ligase reveals the molecular basis for catalytic priming.

Nat Struct Mol Biol 2013 Jun 5;20(6):696-701. Epub 2013 May 5.

Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy.

Homologous to E6-AP C terminus (HECT) E3 ligases recognize and directly catalyze ligation of ubiquitin (Ub) to their substrates. Molecular details of this process remain unknown. We report the first structure, to our knowledge, of a Ub-loaded E3, the human neural precursor cell-expressed developmentally downregulated protein 4 (Nedd4). The HECT(Nedd4)~Ub transitory intermediate provides a structural basis for the proposed sequential addition mechanism. The donor Ub, transferred from the E2, is bound to the Nedd4 C lobe with its C-terminal tail locked in an extended conformation, primed for catalysis. We provide evidence that the Nedd4-family members are Lys63-specific enzymes whose catalysis is mediated by an essential C-terminal acidic residue.
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http://dx.doi.org/10.1038/nsmb.2566DOI Listing
June 2013

Structure of the HECT:ubiquitin complex and its role in ubiquitin chain elongation.

EMBO Rep 2011 Apr 11;12(4):342-9. Epub 2011 Mar 11.

IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Istituto Europeo di Oncologia, Via Adamello 16, Milan 20139, Italy.

Several mechanisms have been proposed for the synthesis of substrate-linked ubiquitin chains. HECT ligases directly catalyse protein ubiquitination and have been found to non-covalently interact with ubiquitin. We report crystal structures of the Nedd4 HECT domain, alone and in complex with ubiquitin, which show a new binding mode involving two surfaces on ubiquitin and both subdomains of the HECT N-lobe. The structures suggest a model for HECT-to-substrate ubiquitin transfer, in which the growing chain on the substrate is kept close to the catalytic cysteine to promote processivity. Mutational analysis highlights differences between the processes of substrate polyubiquitination and self-ubiquitination.
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http://dx.doi.org/10.1038/embor.2011.21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3077247PMC
April 2011

The Ndc80 loop region facilitates formation of kinetochore attachment to the dynamic microtubule plus end.

Curr Biol 2011 Feb 20;21(3):207-13. Epub 2011 Jan 20.

Wellcome Trust Centre for Gene Regulation & Expression, University of Dundee, Dundee DD1 5EH, UK.

Proper chromosome segregation in mitosis relies on correct kinetochore-microtubule (KT-MT) interactions. The KT initially interacts with the lateral surface of a single MT (lateral attachment) extending from a spindle pole and is subsequently anchored at the plus end of the MT (end-on attachment). The conversion from lateral to end-on attachment is crucial because end-on attachment is more robust and thought to be necessary to sustain KT-MT attachment when tension is applied across sister KTs upon their biorientation. The mechanism for this conversion is still elusive. The Ndc80 complex is an essential component of the KT-MT interface, and here we studied a role of the Ndc80 loop region, a distinct motif looping out from the coiled-coil shaft of the complex, in Saccharomyces cerevisiae. With deletions or mutations of the loop region, the lateral KT-MT attachment occurred normally; however, subsequent conversion to end-on attachment was defective, leading to failure in sister KT biorientation. The Ndc80 loop region was required for Ndc80-Dam1 interaction and KT loading of the Dam1 complex, which in turn supported KT tethering to the dynamic MT plus end. The Ndc80 loop region, therefore, has an important role in the conversion from lateral to end-on attachment, a crucial maturation step of KT-MT interaction.
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http://dx.doi.org/10.1016/j.cub.2010.12.050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3052438PMC
February 2011

The Ndc80 kinetochore complex forms oligomeric arrays along microtubules.

Nature 2010 Oct;467(7317):805-10

Biophysics Graduate Group, University of California, Berkeley, California 94720, USA.

The Ndc80 complex is a key site of regulated kinetochore-microtubule attachment (a process required for cell division), but the molecular mechanism underlying its function remains unknown. Here we present a subnanometre-resolution cryo-electron microscopy reconstruction of the human Ndc80 complex bound to microtubules, sufficient for precise docking of crystal structures of the component proteins. We find that the Ndc80 complex binds the microtubule with a tubulin monomer repeat, recognizing α- and β-tubulin at both intra- and inter-tubulin dimer interfaces in a manner that is sensitive to tubulin conformation. Furthermore, Ndc80 complexes self-associate along protofilaments through interactions mediated by the amino-terminal tail of the NDC80 protein, which is the site of phospho-regulation by Aurora B kinase. The complex's mode of interaction with the microtubule and its oligomerization suggest a mechanism by which Aurora B could regulate the stability of load-bearing kinetochore-microtubule attachments.
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http://dx.doi.org/10.1038/nature09423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2957311PMC
October 2010

The MIS12 complex is a protein interaction hub for outer kinetochore assembly.

J Cell Biol 2010 Sep;190(5):835-52

Department of Experimental Oncology, European Institute of Oncology (IEO), Milan, Italy.

Kinetochores are nucleoprotein assemblies responsible for the attachment of chromosomes to spindle microtubules during mitosis. The KMN network, a crucial constituent of the outer kinetochore, creates an interface that connects microtubules to centromeric chromatin. The NDC80, MIS12, and KNL1 complexes form the core of the KMN network. We recently reported the structural organization of the human NDC80 complex. In this study, we extend our analysis to the human MIS12 complex and show that it has an elongated structure with a long axis of approximately 22 nm. Through biochemical analysis, cross-linking-based methods, and negative-stain electron microscopy, we investigated the reciprocal organization of the subunits of the MIS12 complex and their contacts with the rest of the KMN network. A highlight of our findings is the identification of the NSL1 subunit as a scaffold supporting interactions of the MIS12 complex with the NDC80 and KNL1 complexes. Our analysis has important implications for understanding kinetochore organization in different organisms.
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http://dx.doi.org/10.1083/jcb.201002070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2935574PMC
September 2010

Molecular basis for the dual function of Eps8 on actin dynamics: bundling and capping.

PLoS Biol 2010 Jun 1;8(6):e1000387. Epub 2010 Jun 1.

IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy.

Actin capping and cross-linking proteins regulate the dynamics and architectures of different cellular protrusions. Eps8 is the founding member of a unique family of capping proteins capable of side-binding and bundling actin filaments. However, the structural basis through which Eps8 exerts these functions remains elusive. Here, we combined biochemical, molecular, and genetic approaches with electron microscopy and image analysis to dissect the molecular mechanism responsible for the distinct activities of Eps8. We propose that bundling activity of Eps8 is mainly mediated by a compact four helix bundle, which is contacting three actin subunits along the filament. The capping activity is mainly mediated by a amphipathic helix that binds within the hydrophobic pocket at the barbed ends of actin blocking further addition of actin monomers. Single-point mutagenesis validated these modes of binding, permitting us to dissect Eps8 capping from bundling activity in vitro. We further showed that the capping and bundling activities of Eps8 can be fully dissected in vivo, demonstrating the physiological relevance of the identified Eps8 structural/functional modules. Eps8 controls actin-based motility through its capping activity, while, as a bundler, is essential for proper intestinal morphogenesis of developing Caenorhabditis elegans.
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http://dx.doi.org/10.1371/journal.pbio.1000387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2879411PMC
June 2010

Implications for kinetochore-microtubule attachment from the structure of an engineered Ndc80 complex.

Cell 2008 May;133(3):427-39

Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, I 20139 Milan, Italy.

Kinetochores are proteinaceous assemblies that mediate the interaction of chromosomes with the mitotic spindle. The 180 kDa Ndc80 complex is a direct point of contact between kinetochores and microtubules. Its four subunits contain coiled coils and form an elongated rod structure with functional globular domains at either end. We crystallized an engineered "bonsai" Ndc80 complex containing a shortened rod domain but retaining the globular domains required for kinetochore localization and microtubule binding. The structure reveals a microtubule-binding interface containing a pair of tightly interacting calponin-homology (CH) domains with a previously unknown arrangement. The interaction with microtubules is cooperative and predominantly electrostatic. It involves positive charges in the CH domains and in the N-terminal tail of the Ndc80 subunit and negative charges in tubulin C-terminal tails and is regulated by the Aurora B kinase. We discuss our results with reference to current models of kinetochore-microtubule attachment and centromere organization.
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http://dx.doi.org/10.1016/j.cell.2008.03.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4754795PMC
May 2008

Accumulation of Mad2-Cdc20 complex during spindle checkpoint activation requires binding of open and closed conformers of Mad2 in Saccharomyces cerevisiae.

J Cell Biol 2006 Jul;174(1):39-51

Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy.

The spindle assembly checkpoint (SAC) coordinates mitotic progression with sister chromatid alignment. In mitosis, the checkpoint machinery accumulates at kinetochores, which are scaffolds devoted to microtubule capture. The checkpoint protein Mad2 (mitotic arrest deficient 2) adopts two conformations: open (O-Mad2) and closed (C-Mad2). C-Mad2 forms when Mad2 binds its checkpoint target Cdc20 or its kinetochore receptor Mad1. When unbound to these ligands, Mad2 folds as O-Mad2. In HeLa cells, an essential interaction between C- and O-Mad2 conformers allows Mad1-bound C-Mad2 to recruit cytosolic O-Mad2 to kinetochores. In this study, we show that the interaction of the O and C conformers of Mad2 is conserved in Saccharomyces cerevisiae. MAD2 mutant alleles impaired in this interaction fail to restore the SAC in a mad2 deletion strain. The corresponding mutant proteins bind Mad1 normally, but their ability to bind Cdc20 is dramatically impaired in vivo. Our biochemical and genetic evidence shows that the interaction of O- and C-Mad2 is essential for the SAC and is conserved in evolution.
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http://dx.doi.org/10.1083/jcb.200602109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2064158PMC
July 2006

Crystallographic evidence for substrate-assisted GTP hydrolysis by a small GTP binding protein.

Structure 2005 Apr;13(4):533-40

Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France.

GTP hydrolysis by small GTP binding proteins of the Ras superfamily is a universal reaction that controls multiple cellular regulations. Its enzymic mechanism has been the subject of long-standing debates as to the existence/identity of the general base and the electronic nature of its transition state. Here we report the high-resolution crystal structure of a small GTP binding protein, Rab11, solved in complex with GDP and Pi. Unexpectedly, a Pi oxygen and the GDP-cleaved oxygen are located less than 2.5 A apart, suggesting that they share a proton, likely in the form of a low-barrier hydrogen bond. This implies that the gamma-phosphate of GTP was protonated; hence, that GTP acts as a general base. Furthermore, this interaction should establish at, and stabilize, the transition state. Altogether, we propose a revised model for the GTPase reaction that should reconcile earlier models into a unique substrate-assisted mechanism.
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http://dx.doi.org/10.1016/j.str.2005.01.014DOI Listing
April 2005

The structural GDP/GTP cycle of Rab11 reveals a novel interface involved in the dynamics of recycling endosomes.

J Biol Chem 2004 Mar 29;279(12):11480-8. Epub 2003 Dec 29.

Laboratoire d'Enzymologie et Biochimie Structurales, CNRS UPR 9063, Avenue de la Terrasse, 91198 Gif sur Yvette, France.

The small GTP-binding protein Rab11 is an essential regulator of the dynamics of recycling endosomes. Here we report the crystallographic analysis of the GDP/GTP cycle of human Rab11a, and a structure-based mutagenesis study that identifies a novel mutant phenotype. The crystal structures show that the nucleotide-sensitive switch 1 and 2 regions differ from those of other Rab proteins. In Rab11-GDP, they contribute to a close packed symmetrical dimer, which may associate to membranes in the cell and allow Rab11 to undergo GDP/GTP cycles without recycling to the cytosol. The structure of active Rab11 delineates a three-dimensional site that includes switch 1 and is separate from the site defined by the Rab3/Rabphilin interface. It is proposed to form a novel interface for a Rab11 partner compatible with the simultaneous binding of another partner at the Rabphilin interface. Mutation of Ser(29) to Phe in this epitope resulted in morphological modifications of the recycling compartment that are distinct from those induced by the classical dominant-negative and constitutively active Rab11 mutants. Recycling endosomes condensed in the perinuclear region where they retained recycling transferrin, and they clustered Rab11- and EEA1-positive membranes. Altogether, our study suggests that this mutation impairs a specific subset of Rab11 interactions, possibly those involved in cytoskeleton-based movements driving the slow recycling pathway.
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http://dx.doi.org/10.1074/jbc.M310558200DOI Listing
March 2004

Arf, Arl, Arp and Sar proteins: a family of GTP-binding proteins with a structural device for 'front-back' communication.

EMBO Rep 2002 Nov;3(11):1035-41

Laboratoire d'Enzymologie et Biochimie Structurales, UPR 9063 CNRS, 1 avenue de la Terrasse, 91198 Gif sur Yvette cedex, France.

Arf proteins are important regulators of cellular traffic and the founding members of an expanding family of homologous proteins and genomic sequences. They depart from other small GTP-binding proteins by a unique structural device, which we call the 'interswitch toggle', that implements front-back communication from the N-terminus to the nucleotide binding site. Here we define the sequence and structural determinants that propagate information across the protein and identify them in all of the Arf family proteins other than Arl6 and Arl4/Arl7. The positions of these determinants lead us to propose that Arf family members with the interswitch toggle device are activated by a bipartite mechanism acting on opposite sides of the protein. The presence of this communication device might provide a more useful basis for unifying Arf homologs as a family than do the cellular functions of these proteins, which are mostly unrelated. We review available genomic sequences and functional data from this perspective, and identify a novel subfamily that we call Arl8.
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http://dx.doi.org/10.1093/embo-reports/kvf221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1307594PMC
November 2002

Mechanism of domain closure of Sec7 domains and role in BFA sensitivity.

Biochemistry 2002 Mar;41(11):3605-12

Laboratoire d'Enzymologie et Biochimie Structurales, UPR 9063 CNRS, 1, avenue de la Terrasse, 91198 Gif sur Yvette cedex, France.

Activation of small G proteins of the Arf family is initiated by guanine nucleotide exchange factors whose catalytic Sec7 domain stimulates the dissociation of the tightly bound GDP nucleotide. The exchange reaction involves distinct sequential steps that can be trapped by the noncompetitive inhibitor brefeldin A, by mutation of an invariant catalytic glutamate, or by removal of guanine nucleotides. Arf-GDP retains most characteristics of its GDP-bound form at the initial low-affinity Arf-GDP-Sec7 step. It then undergoes large conformational changes toward its GTP-bound form at the next step, and eventually dissociates GDP to form a nucleotide-free high-affinity Arf-Sec7 complex at the last step. Thus, Arf proteins evolve through different conformations that must be accommodated by Sec7 domains in the course of the reaction. Here the contribution of the flexibility of Sec7 domains to the exchange reaction was investigated with the crystal structure of the unbound Sec7 domain of yeast Gea2. Comparison with Gea2 in complex with nucleotide-free Arf1 Delta 17 [Goldberg, J. (1998) Cell 95, 237-248] reveals that Arf induces closure of the two subdomains that form the sides of its active site. Several residues that determine sensitivity to brefeldin A are involved in interdomain and local movements, pointing to the importance of the flexibility of Sec7 domains for the inhibition mechanism. Altogether, this suggests a model for the initial steps of the exchange reaction where Arf docks onto the C-terminal domain of the Sec7 domain before closure of the N-terminal domain positions the catalytic glutamate to complete the reaction.
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http://dx.doi.org/10.1021/bi012123hDOI Listing
March 2002