Publications by authors named "Michel O Steinmetz"

145 Publications

Centrosomes in the spotlight: from organization to function to role in disease.

Curr Opin Struct Biol 2021 Feb 20;66:iii-v. Epub 2021 Jan 20.

Laboratory of Biomolecular Research, Division of Biology and Chemistry, Forschungsstrasse 111, CH-5232 Villigen PSI, Switzerland. Electronic address:

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http://dx.doi.org/10.1016/j.sbi.2021.01.001DOI Listing
February 2021

Surface tensiometry of phase separated protein and polymer droplets by the sessile drop method.

Soft Matter 2021 Feb;17(6):1655-1662

Department of Materials, ETH Zürich, Switzerland.

Phase separated macromolecules play essential roles in many biological and synthetic systems. Physical characterization of these systems can be challenging because of limited sample volumes, particularly for phase-separated proteins. Here, we demonstrate that a classic method for measuring the surface tension of liquid droplets, based on the analysis of the shape of a sessile droplet, can be effectively scaled down to measure the interfacial tension between a macromolecule-rich droplet phase and its co-existing macromolecule-poor continuous phase. The connection between droplet shape and surface tension relies on the density difference between the droplet and its surroundings. This can be determined with small sample volumes in the same setup by measuring the droplet sedimentation velocity. An interactive MATLAB script for extracting the capillary length from a droplet image is included in the ESI.
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http://dx.doi.org/10.1039/d0sm01319fDOI Listing
February 2021

A Robust, GFP-Orthogonal Photoswitchable Inhibitor Scaffold Extends Optical Control over the Microtubule Cytoskeleton.

Cell Chem Biol 2021 Feb 3;28(2):228-241.e6. Epub 2020 Dec 3.

Department of Pharmacy, Ludwig-Maximilians University of Munich, Munich 81377, Germany. Electronic address:

Optically controlled chemical reagents, termed "photopharmaceuticals," are powerful tools for precise spatiotemporal control of proteins particularly when genetic methods, such as knockouts or optogenetics are not viable options. However, current photopharmaceutical scaffolds, such as azobenzenes are intolerant of GFP/YFP imaging and are metabolically labile, posing severe limitations for biological use. We rationally designed a photoswitchable "SBT" scaffold to overcome these problems, then derivatized it to create exceptionally metabolically robust and fully GFP/YFP-orthogonal "SBTub" photopharmaceutical tubulin inhibitors. Lead compound SBTub3 allows temporally reversible, cell-precise, and even subcellularly precise photomodulation of microtubule dynamics, organization, and microtubule-dependent processes. By overcoming the previous limitations of microtubule photopharmaceuticals, SBTubs offer powerful applications in cell biology, and their robustness and druglikeness are favorable for intracellular biological control in in vivo applications. We furthermore expect that the robustness and imaging orthogonality of the SBT scaffold will inspire other derivatizations directed at extending the photocontrol of a range of other biological targets.
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http://dx.doi.org/10.1016/j.chembiol.2020.11.007DOI Listing
February 2021

The mechanism of kinesin inhibition by kinesin-binding protein.

Elife 2020 11 30;9. Epub 2020 Nov 30.

Institute of Structural and Molecular Biology, Birkbeck, University of London, London, United Kingdom.

Subcellular compartmentalisation is necessary for eukaryotic cell function. Spatial and temporal regulation of kinesin activity is essential for building these local environments via control of intracellular cargo distribution. Kinesin-binding protein (KBP) interacts with a subset of kinesins via their motor domains, inhibits their microtubule (MT) attachment, and blocks their cellular function. However, its mechanisms of inhibition and selectivity have been unclear. Here we use cryo-electron microscopy to reveal the structure of KBP and of a KBP-kinesin motor domain complex. KBP is a tetratricopeptide repeat-containing, right-handed α-solenoid that sequesters the kinesin motor domain's tubulin-binding surface, structurally distorting the motor domain and sterically blocking its MT attachment. KBP uses its α-solenoid concave face and edge loops to bind the kinesin motor domain, and selected structure-guided mutations disrupt KBP inhibition of kinesin transport in cells. The KBP-interacting motor domain surface contains motifs exclusively conserved in KBP-interacting kinesins, suggesting a basis for kinesin selectivity.
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http://dx.doi.org/10.7554/eLife.61481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7746232PMC
November 2020

Centriole length control.

Curr Opin Struct Biol 2021 Feb 18;66:89-95. Epub 2020 Nov 18.

Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institut, CH-5232 Villigen, Switzerland; University of Basel, Biozentrum, CH-4056 Basel, Switzerland. Electronic address:

Centrioles are microtubule-based structures involved in cell division and ciliogenesis. Centriole formation is a highly regulated cellular process and aberrations in centriole structure, size or numbers have implications in multiple human pathologies. In this review, we propose that the proteins that control centriole length can be subdivided into two classes based on their antagonistic activities on centriolar microtubules, which we refer to as 'centriole elongation activators' (CEAs) and 'centriole elongation inhibitors' (CEIs). We discuss and illustrate the structure-function relationship of CEAs and CEIs as well as their interaction networks. Based on our current knowledge, we formulate some outstanding open questions in the field and present possible routes for future studies.
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http://dx.doi.org/10.1016/j.sbi.2020.10.011DOI Listing
February 2021

Advances in long-wavelength native phasing at X-ray free-electron lasers.

IUCrJ 2020 Nov 9;7(Pt 6):965-975. Epub 2020 Sep 9.

Photon Science Division, Paul Scherrer Institut, Forschungsstrasse 111, Villigen PSI, 5232, Switzerland.

Long-wavelength pulses from the Swiss X-ray free-electron laser (XFEL) have been used for protein structure determination by native single-wavelength anomalous diffraction (native-SAD) phasing of serial femtosecond crystallography (SFX) data. In this work, sensitive anomalous data-quality indicators and model proteins were used to quantify improvements in native-SAD at XFELs such as utilization of longer wavelengths, careful experimental geometry optimization, and better post-refinement and partiality correction. Compared with studies using shorter wavelengths at other XFELs and older software versions, up to one order of magnitude reduction in the required number of indexed images for native-SAD was achieved, hence lowering sample consumption and beam-time requirements significantly. Improved data quality and higher anomalous signal facilitate so-far underutilized structure determination of challenging proteins at XFELs. Improvements presented in this work can be used in other types of SFX experiments that require accurate measurements of weak signals, for example time-resolved studies.
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http://dx.doi.org/10.1107/S2052252520011379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642782PMC
November 2020

WDR90 is a centriolar microtubule wall protein important for centriole architecture integrity.

Elife 2020 09 18;9. Epub 2020 Sep 18.

University of Geneva, Department of Cell Biology, Sciences III, Geneva, Switzerland.

Centrioles are characterized by a nine-fold arrangement of microtubule triplets held together by an inner protein scaffold. These structurally robust organelles experience strenuous cellular processes such as cell division or ciliary beating while performing their function. However, the molecular mechanisms underlying the stability of microtubule triplets, as well as centriole architectural integrity remain poorly understood. Here, using ultrastructure expansion microscopy for nanoscale protein mapping, we reveal that POC16 and its human homolog WDR90 are components of the microtubule wall along the central core region of the centriole. We further found that WDR90 is an evolutionary microtubule associated protein. Finally, we demonstrate that WDR90 depletion impairs the localization of inner scaffold components, leading to centriole structural abnormalities in human cells. Altogether, this work highlights that WDR90 is an evolutionary conserved molecular player participating in centriole architecture integrity.
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http://dx.doi.org/10.7554/eLife.57205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7500955PMC
September 2020

Structural Basis of Noscapine Activation for Tubulin Binding.

J Med Chem 2020 08 29;63(15):8495-8501. Epub 2020 Jul 29.

Centro de Investigaciones Biológicas Margarita Salas-CSIC, Ramiro de Maeztu, 9, C.P. 28040 Madrid, Spain.

Noscapine is a natural alkaloid that is used as an antitussive medicine. However, it also acts as a weak anticancer agent in certain models through a mechanism that is largely unknown. Here, we performed structural studies and show that the cytotoxic agent 7A--demethoxy-amino-noscapine (7A-aminonoscapine) binds to the colchicine site of tubulin. We suggest that the 7A-methoxy group of noscapine prevents binding to tubulin due to a steric clash of the compound with the T5-loop of α-tubulin. We further propose that the anticancer activity of noscapine arises from a bioactive metabolite that binds to the colchicine site of tubulin to induce mitotic arrest through a microtubule cytoskeleton-based mechanism.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00855DOI Listing
August 2020

Pharmaceutical-Grade Rigosertib Is a Microtubule-Destabilizing Agent.

Mol Cell 2020 07;79(1):191-198.e3

Department of Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address:

We recently used CRISPRi/a-based chemical-genetic screens and cell biological, biochemical, and structural assays to determine that rigosertib, an anti-cancer agent in phase III clinical trials, kills cancer cells by destabilizing microtubules. Reddy and co-workers (Baker et al., 2020, this issue of Molecular Cell) suggest that a contaminating degradation product in commercial formulations of rigosertib is responsible for the microtubule-destabilizing activity. Here, we demonstrate that cells treated with pharmaceutical-grade rigosertib (>99.9% purity) or commercially obtained rigosertib have qualitatively indistinguishable phenotypes across multiple assays. The two formulations have indistinguishable chemical-genetic interactions with genes that modulate microtubule stability, both destabilize microtubules in cells and in vitro, and expression of a rationally designed tubulin mutant with a mutation in the rigosertib binding site (L240F TUBB) allows cells to proliferate in the presence of either formulation. Importantly, the specificity of the L240F TUBB mutant for microtubule-destabilizing agents has been confirmed independently. Thus, rigosertib kills cancer cells by destabilizing microtubules, in agreement with our original findings.
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http://dx.doi.org/10.1016/j.molcel.2020.06.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7332992PMC
July 2020

Structural Refinement of the Tubulin Ligand (+)-Discodermolide to Attenuate Chemotherapy-Mediated Senescence.

Mol Pharmacol 2020 08 26;98(2):156-167. Epub 2020 Jun 26.

Department of Chemistry, Monell Chemical Senses Center and Laboratory for Research on the Structure of Matter, University of Pennsylvania, Philadelphia, Pennsylvania (B.G., N.Z., A.B.S.); Departments of Molecular Pharmacology (A.R.-G., S.B.H., H.M.M.), Epidemiology (K.Y.), and Medicine (H.M.M.), Albert Einstein College of Medicine, Bronx, New York; Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institut, Villigen, Switzerland (A.E.P., T.M., M.O.S.); and University of Basel, Biozentrum, Basel, Switzerland (M.O.S.)

The natural product (+)-discodermolide (DDM) is a microtubule stabilizing agent and potent inducer of senescence. We refined the structure of DDM and evaluated the activity of novel congeners in triple negative breast and ovarian cancers, malignancies that typically succumb to taxane resistance. Previous structure-activity analyses identified the lactone and diene as moieties conferring anticancer activity, thus identifying priorities for the structural refinement studies described herein. Congeners possessing the monodiene with a simplified lactone had superior anticancer efficacy relative to taxol, particularly in resistant models. Specifically, one of these congeners, B2, demonstrated 1) improved pharmacologic properties, specifically increased maximum response achievable and area under the curve, and decreased EC; 2) a uniform dose-response profile across genetically heterogeneous cancer cell lines relative to taxol or DDM; 3) reduced propensity for senescence induction relative to DDM; 4) superior long-term activity in cancer cells versus taxol or DDM; and 5) attenuation of metastatic characteristics in treated cancer cells. To contrast the binding of B2 versus DDM in tubulin, X-ray crystallography studies revealed a shift in the position of the lactone ring associated with removal of the C2-methyl and C3-hydroxyl. Thus, B2 may be more adaptable to changes in the taxane site relative to DDM that could account for its favorable properties. In conclusion, we have identified a DDM congener with broad range anticancer efficacy that also has decreased risk of inducing chemotherapy-mediated senescence. SIGNIFICANCE STATEMENT: Here, we describe the anticancer activity of novel congeners of the tubulin-polymerizing molecule (+)-discodermolide. A lead molecule is identified that exhibits an improved dose-response profile in taxane-sensitive and taxane-resistant cancer cell models, diminished risk of chemotherapy-mediated senescence, and suppression of tumor cell invasion endpoints. X-ray crystallography studies identify subtle changes in the pose of binding to -tubulin that could account for the improved anticancer activity. These findings support continued preclinical development of discodermolide, particularly in the chemorefractory setting.
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http://dx.doi.org/10.1124/mol.119.117457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7362599PMC
August 2020

Author Correction: A structural model for microtubule minus-end recognition and protection by CAMSAP proteins.

Nat Struct Mol Biol 2020 Jun;27(6):603

Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Utrecht, the Netherlands.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41594-020-0382-zDOI Listing
June 2020

Homodimerization of coronin A through the C-terminal coiled-coil domain is essential for multicellular differentiation of Dictyostelium discoideum.

FEBS Lett 2020 Apr 16. Epub 2020 Apr 16.

Biozentrum, University of Basel, Switzerland.

Coronin proteins are widely expressed among eukaryotic organisms. Most coronins consist of a WD-repeat domain followed by a C-terminal coiled coil. Dictyostelium discoideum expresses a single short coronin coronin A, which has been implicated in both actin modulation and multicellular differentiation. Whether coronin A's coiled coil is important for functionality, as well as the oligomeric state of coronin A is not known. Here, we show that the coiled-coil domain in Dictyostelium coronin A functions in homodimerization, is dispensable for coronin A stability and localization but essential for multicellular differentiation. These results allow a better understanding of the role for the coiled-coil domain of coronin A in oligomerization and demonstrate that its presence is essential for multicellular differentiation.
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http://dx.doi.org/10.1002/1873-3468.13787DOI Listing
April 2020

Structural model for differential cap maturation at growing microtubule ends.

Elife 2020 03 10;9. Epub 2020 Mar 10.

Structural and Chemical Biology Department, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.

Microtubules (MTs) are hollow cylinders made of tubulin, a GTPase responsible for essential functions during cell growth and division, and thus, key target for anti-tumor drugs. In MTs, GTP hydrolysis triggers structural changes in the lattice, which are responsible for interaction with regulatory factors. The stabilizing GTP-cap is a hallmark of MTs and the mechanism of the chemical-structural link between the GTP hydrolysis site and the MT lattice is a matter of debate. We have analyzed the structure of tubulin and MTs assembled in the presence of fluoride salts that mimic the GTP-bound and GDP•P transition states. Our results challenge current models because tubulin does not change axial length upon GTP hydrolysis. Moreover, analysis of the structure of MTs assembled in the presence of several nucleotide analogues and of taxol allows us to propose that previously described lattice expansion could be a post-hydrolysis stage involved in P release.
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http://dx.doi.org/10.7554/eLife.50155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064335PMC
March 2020

Mechanisms of Motor-Independent Membrane Remodeling Driven by Dynamic Microtubules.

Curr Biol 2020 03 7;30(6):972-987.e12. Epub 2020 Feb 7.

Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, Utrecht 3584, the Netherlands. Electronic address:

Microtubule-dependent organization of membranous organelles occurs through motor-based pulling and by coupling microtubule dynamics to membrane remodeling. For example, tubules of endoplasmic reticulum (ER) can be extended by kinesin- and dynein-mediated transport and through the association with the tips of dynamic microtubules. The binding between ER and growing microtubule plus ends requires End Binding (EB) proteins and the transmembrane protein STIM1, which form a tip-attachment complex (TAC), but it is unknown whether these proteins are sufficient for membrane remodeling. Furthermore, EBs and their partners undergo rapid turnover at microtubule ends, and it is unclear how highly transient protein-protein interactions can induce load-bearing processive motion. Here, we reconstituted membrane tubulation in a minimal system with giant unilamellar vesicles, dynamic microtubules, an EB protein, and a membrane-bound protein that can interact with EBs and microtubules. We showed that these components are sufficient to drive membrane remodeling by three mechanisms: membrane tubulation induced by growing microtubule ends, motor-independent membrane sliding along microtubule shafts, and membrane pulling by shrinking microtubules. Experiments and modeling demonstrated that the first two mechanisms can be explained by adhesion-driven biased membrane spreading on microtubules. Optical trapping revealed that growing and shrinking microtubule ends can exert forces of ∼0.5 and ∼5 pN, respectively, through attached proteins. Rapidly exchanging molecules that connect membranes to dynamic microtubules can thus bear a sufficient load to induce membrane deformation and motility. Furthermore, combining TAC components and a membrane-attached kinesin in the same in vitro assays demonstrated that they can cooperate in promoting membrane tubule extension.
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http://dx.doi.org/10.1016/j.cub.2020.01.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7090928PMC
March 2020

Taxanes convert regions of perturbed microtubule growth into rescue sites.

Nat Mater 2020 03 9;19(3):355-365. Epub 2019 Dec 9.

Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht, the Netherlands.

Microtubules are polymers of tubulin dimers, and conformational transitions in the microtubule lattice drive microtubule dynamic instability and affect various aspects of microtubule function. The exact nature of these transitions and their modulation by anticancer drugs such as Taxol and epothilone, which can stabilize microtubules but also perturb their growth, are poorly understood. Here, we directly visualize the action of fluorescent Taxol and epothilone derivatives and show that microtubules can transition to a state that triggers cooperative drug binding to form regions with altered lattice conformation. Such regions emerge at growing microtubule ends that are in a pre-catastrophe state, and inhibit microtubule growth and shortening. Electron microscopy and in vitro dynamics data indicate that taxane accumulation zones represent incomplete tubes that can persist, incorporate tubulin dimers and repeatedly induce microtubule rescues. Thus, taxanes modulate the material properties of microtubules by converting destabilized growing microtubule ends into regions resistant to depolymerization.
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http://dx.doi.org/10.1038/s41563-019-0546-6DOI Listing
March 2020

Structural determinants of microtubule minus end preference in CAMSAP CKK domains.

Nat Commun 2019 11 20;10(1):5236. Epub 2019 Nov 20.

Institute of Structural and Molecular Biology, Birkbeck, University of London, Malet Street, London, UK.

CAMSAP/Patronins regulate microtubule minus-end dynamics. Their end specificity is mediated by their CKK domains, which we proposed recognise specific tubulin conformations found at minus ends. To critically test this idea, we compared the human CAMSAP1 CKK domain (HsCKK) with a CKK domain from Naegleria gruberi (NgCKK), which lacks minus-end specificity. Here we report near-atomic cryo-electron microscopy structures of HsCKK- and NgCKK-microtubule complexes, which show that these CKK domains share the same protein fold, bind at the intradimer interprotofilament tubulin junction, but exhibit different footprints on microtubules. NMR experiments show that both HsCKK and NgCKK are remarkably rigid. However, whereas NgCKK binding does not alter the microtubule architecture, HsCKK remodels its microtubule interaction site and changes the underlying polymer structure because the tubulin lattice conformation is not optimal for its binding. Thus, in contrast to many MAPs, the HsCKK domain can differentiate subtly specific tubulin conformations to enable microtubule minus-end recognition.
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http://dx.doi.org/10.1038/s41467-019-13247-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868217PMC
November 2019

GEF-H1 Signaling upon Microtubule Destabilization Is Required for Dendritic Cell Activation and Specific Anti-tumor Responses.

Cell Rep 2019 09;28(13):3367-3380.e8

Department of Biomedicine, University Hospital Basel and University of Basel, 4031 Basel, Switzerland; Medical Oncology, University Hospital Basel, 4031 Basel, Switzerland. Electronic address:

Dendritic cell (DC) activation is a critical step for anti-tumor T cell responses. Certain chemotherapeutics can influence DC function. Here we demonstrate that chemotherapy capable of microtubule destabilization has direct effects on DC function; namely, it induces potent DC maturation and elicits anti-tumor immunity. Guanine nucleotide exchange factor-H1 (GEF-H1) is specifically released upon microtubule destabilization and is required for DC activation. In response to chemotherapy, GEF-H1 drives a distinct cell signaling program in DCs dominated by the c-Jun N-terminal kinase (JNK) pathway and AP-1/ATF transcriptional response for control of innate and adaptive immune responses. Microtubule destabilization, and subsequent GEF-H1 signaling, enhances cross-presentation of tumor antigens to CD8 T cells. In absence of GEF-H1, anti-tumor immunity is hampered. In cancer patients, high expression of the GEF-H1 immune gene signature is associated with prolonged survival. Our study identifies an alternate intracellular axis in DCs induced upon microtubule destabilization in which GEF-H1 promotes protective anti-tumor immunity.
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http://dx.doi.org/10.1016/j.celrep.2019.08.057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6876861PMC
September 2019

Remote control of microtubule plus-end dynamics and function from the minus-end.

Elife 2019 09 6;8. Epub 2019 Sep 6.

Institute of Biochemistry, ETH Zürich, Zurich, Switzerland.

In eukaryotes, the organization and function of the microtubule cytoskeleton depend on the allocation of different roles to individual microtubules. For example, many asymmetrically dividing cells differentially specify microtubule behavior at old and new centrosomes. Here we show that yeast spindle pole bodies (SPBs, yeast centrosomes) differentially control the plus-end dynamics and cargoes of their astral microtubules, remotely from the minus-end. The old SPB recruits the kinesin motor protein Kip2, which then translocates to the plus-end of the emanating microtubules, promotes their extension and delivers dynein into the bud. Kip2 recruitment at the SPB depends on Bub2 and Bfa1, and phosphorylation of cytoplasmic Kip2 prevents random lattice binding. Releasing Kip2 of its control by SPBs equalizes its distribution, the length of microtubules and dynein distribution between the mother cell and its bud. These observations reveal that microtubule organizing centers use minus to plus-end directed remote control to individualize microtubule function.
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http://dx.doi.org/10.7554/eLife.48627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754230PMC
September 2019

Crystal Structure of a Heterotetrameric Katanin p60:p80 Complex.

Structure 2019 09 25;27(9):1375-1383.e3. Epub 2019 Jul 25.

Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institut, 5232 Villigen PSI, Switzerland. Electronic address:

Katanin is a microtubule-severing enzyme that is crucial for many cellular processes. Katanin consists of two subunits, p60 and p80, that form a stable complex. The interaction between subunits is mediated by the p60 N-terminal microtubule-interacting and -trafficking domain (p60-MIT) and the p80 C-terminal domain (p80-CTD). Here, we performed a biophysical characterization of the mouse p60-MIT:p80-CTD heterodimer and show that this complex can assemble into heterotetramers. We identified two mutations that enhance heterotetramer formation and determined the X-ray crystal structure of this mutant complex. The structure revealed a domain-swapped heterotetramer consisting of two p60-MIT:p80-CTD heterodimers. Structure-based sequence alignments suggest that heterotetramerization of katanin might be a common feature of various species. Furthermore, we show that enhanced heterotetramerization of katanin impairs its microtubule end-binding properties and increases the enzyme's microtubule lattice binding and severing activities. Therefore, our findings suggest the existence of different katanin oligomers that possess distinct functional properties.
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http://dx.doi.org/10.1016/j.str.2019.07.002DOI Listing
September 2019

VISAGE Reveals a Targetable Mitotic Spindle Vulnerability in Cancer Cells.

Cell Syst 2019 07 10;9(1):74-92.e8. Epub 2019 Jul 10.

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Center for Precision Cancer Medicine, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address:

There is an unmet need for new antimitotic drug combinations that target cancer-specific vulnerabilities. Based on our finding of elevated biomolecule oxidation in mitotically arrested cancer cells, we combined Plk1 inhibitors with TH588, an MTH1 inhibitor that prevents detoxification of oxidized nucleotide triphosphates. This combination showed robust synergistic killing of cancer, but not normal, cells that, surprisingly, was MTH1-independent. To dissect the underlying synergistic mechanism, we developed VISAGE, a strategy integrating experimental synergy quantification with computational-pathway-based gene expression analysis. VISAGE predicted, and we experimentally confirmed, that this synergistic combination treatment targeted the mitotic spindle. Specifically, TH588 binding to β-tubulin impaired microtubule assembly, which when combined with Plk1 blockade, synergistically disrupted mitotic chromosome positioning to the spindle midzone. These findings identify a cancer-specific mitotic vulnerability that is targetable using Plk1 inhibitors with microtubule-destabilizing agents and highlight the general utility of the VISAGE approach to elucidate molecular mechanisms of drug synergy.
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http://dx.doi.org/10.1016/j.cels.2019.05.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688637PMC
July 2019

Structural basis of tubulin detyrosination by the vasohibin-SVBP enzyme complex.

Nat Struct Mol Biol 2019 07 24;26(7):571-582. Epub 2019 Jun 24.

Department of Biology, Southern University of Science and Technology, Shenzhen, China.

Vasohibins are tubulin tyrosine carboxypeptidases that are important in neuron physiology. We examined the crystal structures of human vasohibin 1 and 2 in complex with small vasohibin-binding protein (SVBP) in the absence and presence of different inhibitors and a C-terminal α-tubulin peptide. In combination with functional data, we propose that SVBP acts as an activator of vasohibins. An extended groove and a distinctive surface residue patch of vasohibins define the specific determinants for recognizing and cleaving the C-terminal tyrosine of α-tubulin and for binding microtubules, respectively. The vasohibin-SVBP interaction and the ability of the enzyme complex to associate with microtubules regulate axon specification of neurons. Our results define the structural basis of tubulin detyrosination by vasohibins and show the relevance of this process for neuronal development. Our findings offer a unique platform for developing drugs against human conditions with abnormal tubulin tyrosination levels, such as cancer, heart defects and possibly brain disorders.
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http://dx.doi.org/10.1038/s41594-019-0241-yDOI Listing
July 2019

Microtubule minus-end regulation at a glance.

J Cell Sci 2019 06 7;132(11). Epub 2019 Jun 7.

Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland

Microtubules are cytoskeletal filaments essential for numerous aspects of cell physiology. They are polarized polymeric tubes with a fast growing plus end and a slow growing minus end. In this Cell Science at a Glance article and the accompanying poster, we review the current knowledge on the dynamics and organization of microtubule minus ends. Several factors, including the γ-tubulin ring complex, CAMSAP/Patronin, ASPM/Asp, SPIRAL2 (in plants) and the KANSL complex recognize microtubule minus ends and regulate their nucleation, stability and interactions with partners, such as microtubule severing enzymes, microtubule depolymerases and protein scaffolds. Together with minus-end-directed motors, these microtubule minus-end targeting proteins (-TIPs) also control the formation of microtubule-organizing centers, such as centrosomes and spindle poles, and mediate microtubule attachment to cellular membrane structures, including the cell cortex, Golgi complex and the cell nucleus. Structural and functional studies are starting to reveal the molecular mechanisms by which dynamic -TIP networks control microtubule minus ends.
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http://dx.doi.org/10.1242/jcs.227850DOI Listing
June 2019

Crystal Structure of the Cyclostreptin-Tubulin Adduct: Implications for Tubulin Activation by Taxane-Site Ligands.

Int J Mol Sci 2019 Mar 20;20(6). Epub 2019 Mar 20.

Structural and Chemical Biology Department. Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.

It has been proposed that one of the mechanisms of taxane-site ligand-mediated tubulin activation is modulation of the structure of a switch element (the M-loop) from a disordered form in dimeric tubulin to a folded helical structure in microtubules. Here, we used covalent taxane-site ligands, including cyclostreptin, to gain further insight into this mechanism. The crystal structure of cyclostreptin-bound tubulin reveals covalent binding to βHis229, but no stabilization of the M-loop. The capacity of cyclostreptin to induce microtubule assembly compared to other covalent taxane-site agents demonstrates that the induction of tubulin assembly is not strictly dependent on M-loop stabilization. We further demonstrate that most covalent taxane-site ligands are able to partially overcome drug resistance mediated by βIII-tubulin (βIII) overexpression in HeLa cells, and compare their activities to pironetin, an interfacial covalent inhibitor of tubulin assembly that displays invariant growth inhibition in these cells. Our findings suggest a relationship between a diminished interaction of taxane-site ligands with βIII-tubulin and βIII tubulin-mediated drug resistance. This supports the idea that overexpression of βIII increases microtubule dynamicity by counteracting the enhanced microtubule stability promoted by covalent taxane-site binding ligands.
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http://dx.doi.org/10.3390/ijms20061392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6471726PMC
March 2019

Conformational Properties of the Chemotherapeutic Drug Analogue Epothilone A: How to Model a Flexible Protein Ligand Using Scarcely Available Experimental Data.

J Chem Inf Model 2019 05 25;59(5):2218-2230. Epub 2019 Mar 25.

Laboratory of Biomolecular Research, Division of Biology and Chemistry , Paul Scherrer Institut , CH-5232 Villigen , Switzerland.

Epothilones are among the most potent chemotherapeutic drugs used for the treatment of cancer. Epothilone A (EpoA), a natural product, is a macrocyclic molecule containing 34 non-hydrogen atoms and a thiazole side chain. NMR studies of EpoA in aqueous solution, unbound as well as bound to αβ-tubulin, and unbound in dimethyl sulfoxide (DMSO) solution have delivered sets of nuclear Overhauser effect (NOE) atom-atom distance bounds, but no structures based on NMR data are present in structural data banks. X-ray diffraction of crystals has provided structures of EpoA unbound and bound to αβ-tubulin. Since both crystal structures derived from X-ray diffraction intensities do not completely satisfy the three available sets of NOE distance bounds for EpoA, molecular dynamics (MD) simulations have been employed to obtain conformational ensembles in aqueous and in DMSO solution that are compatible with the respective NOE data. It was found that EpoA displays a larger conformational variability in DMSO than in water and the two conformational ensembles show little overlap. Yet, they both provide conformational scaffolds that are energetically accessible at physiological temperature and pressure.
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http://dx.doi.org/10.1021/acs.jcim.9b00171DOI Listing
May 2019

Tetrahydroisoquinoline Sulfamates as Potent Microtubule Disruptors: Synthesis, Antiproliferative and Antitubulin Activity of Dichlorobenzyl-Based Derivatives, and a Tubulin Cocrystal Structure.

ACS Omega 2019 Jan 9;4(1):755-764. Epub 2019 Jan 9.

Medicinal Chemistry & Drug Discovery, Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, U.K.

Tetrahydroisoquinoline (THIQ) 6--sulfamate-based anticancer agents, inspired by the endogenous steroid 2-methoxyestradiol and its sulfamate derivatives, are further explored for antiproliferative and microtubule disruptor activity. Based on recently designed C3-methyl C7-methoxy-substituted THIQ derivatives, compounds with mono- and dichloro-substitutions on the pendant -benzyl ring were synthesized and evaluated. Although improved antiproliferative activity was observed, for example, versus and versus , it was relatively modest. Compound , a 2',5'-dichlorobenzyl derivative was, however, identified as a promising antiproliferative agent with in vitro activities exceeding that of the parent steroid (e.g., GI 90 nM in DU-145 cells) and was highly potent against a range of tumor cell lines (e.g., GI 26 nM for OVCAR-3). inhibited the polymerization of tubulin in vitro with an IC only twofold less potent than combretastatin A-4 and inhibited colchicine binding to tubulin. Tubulin polymerization assays showed the parent THIQ to be only a very weak inhibitor, but a striking potency difference was seen between compounds with C2' methoxy and chloro substituents, whereas this was much smaller when these substituents were positioned at C5'. To confirm the target in atomic detail and because is a racemic mixture, an achiral parent THIQ 6--sulfamate derivative was successfully cocrystallized with the αβ-tubulin heterodimer. The derivative binds at the colchicine site on tubulin, the first example of this compound class investigated in such detail, with its sulfamate group interacting with residues beyond the reach of colchicine itself, similar to a recently reported quinazolinone sulfamate derivative, . The structure also suggests that for racemic C3-methyl-substituted THIQ derivatives, such as , the (S)-enantiomer is likely to be preferentially accommodated within the colchicine site for steric reasons. The results further confirm the potential of nonsteroidal THIQ sulfamate derivatives for oncology and suggest that the mechanism of microtubule destabilization for the THIQ compound class is to prevent the curved-to-straight conformational transition of tubulin required for polymerization.
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http://dx.doi.org/10.1021/acsomega.8b02879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6372245PMC
January 2019

MAP7 family proteins regulate kinesin-1 recruitment and activation.

J Cell Biol 2019 04 15;218(4):1298-1318. Epub 2019 Feb 15.

Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Utrecht, Netherlands

Kinesin-1 is responsible for microtubule-based transport of numerous cellular cargoes. Here, we explored the regulation of kinesin-1 by MAP7 proteins. We found that all four mammalian MAP7 family members bind to kinesin-1. In HeLa cells, MAP7, MAP7D1, and MAP7D3 act redundantly to enable kinesin-1-dependent transport and microtubule recruitment of the truncated kinesin-1 KIF5B-560, which contains the stalk but not the cargo-binding and autoregulatory regions. In vitro, purified MAP7 and MAP7D3 increase microtubule landing rate and processivity of kinesin-1 through transient association with the motor. MAP7 proteins promote binding of kinesin-1 to microtubules both directly, through the N-terminal microtubule-binding domain and unstructured linker region, and indirectly, through an allosteric effect exerted by the kinesin-binding C-terminal domain. Compared with MAP7, MAP7D3 has a higher affinity for kinesin-1 and a lower affinity for microtubules and, unlike MAP7, can be cotransported with the motor. We propose that MAP7 proteins are microtubule-tethered kinesin-1 activators, with which the motor transiently interacts as it moves along microtubules.
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http://dx.doi.org/10.1083/jcb.201808065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446838PMC
April 2019

Structure-activity relationships, biological evaluation and structural studies of novel pyrrolonaphthoxazepines as antitumor agents.

Eur J Med Chem 2019 Jan 3;162:290-320. Epub 2018 Nov 3.

European Research Centre for Drug Discovery and Development (NatSynDrugs), University of Siena, Department of Biotechnology, Chemistry and Pharmacy, DoE Department of Excellence 2018-2022, via Aldo Moro 2, I-53100, Siena, Italy; Istituto Toscano Tumori, University of Siena, via Aldo Moro 2, I-53100, Siena, Italy; Department of Pharmacy, University of Napoli Federico II, DoE Department of Excellence 2018-2022, Via D. Montesano 49, 80131, Napoli, Italy.

Microtubule-targeting agents (MTAs) are a class of clinically successful anti-cancer drugs. The emergence of multidrug resistance to MTAs imposes the need for developing new MTAs endowed with diverse mechanistic properties. Benzoxazepines were recently identified as a novel class of MTAs. These anticancer agents were thoroughly characterized for their antitumor activity, although, their exact mechanism of action remained elusive. Combining chemical, biochemical, cellular, bioinformatics and structural efforts we developed improved pyrrolonaphthoxazepines antitumor agents and their mode of action at the molecular level was elucidated. Compound 6j, one of the most potent analogues, was confirmed by X-ray as a colchicine-site MTA. A comprehensive structural investigation was performed for a complete elucidation of the structure-activity relationships. Selected pyrrolonaphthoxazepines were evaluated for their effects on cell cycle, apoptosis and differentiation in a variety of cancer cells, including multidrug resistant cell lines. Our results define compound 6j as a potentially useful optimized hit for the development of effective compounds for treating drug-resistant tumors.
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http://dx.doi.org/10.1016/j.ejmech.2018.11.004DOI Listing
January 2019

Combinatorial use of disulfide bridges and native sulfur-SAD phasing for rapid structure determination of coiled-coils.

Biosci Rep 2018 10 19;38(5). Epub 2018 Sep 19.

Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland

Coiled-coils are ubiquitous protein-protein interaction motifs found in many eukaryotic proteins. The elongated, flexible and often irregular nature of coiled-coils together with their tendency to form fibrous arrangements in crystals imposes challenges on solving the phase problem by molecular replacement. Here, we report the successful combinatorial use of native and rational engineered disulfide bridges together with sulfur-SAD phasing as a powerful tool to stabilize and solve the structure of coiled-coil domains in a straightforward manner. Our study is a key example of how modern sulfur SAD combined with mutagenesis can help to advance and simplify the structural study of challenging coiled-coil domains by X-ray crystallography.
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http://dx.doi.org/10.1042/BSR20181073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6146289PMC
October 2018

Sustainable Syntheses of (-)-Jerantinines A & E and Structural Characterisation of the Jerantinine-Tubulin Complex at the Colchicine Binding Site.

Sci Rep 2018 Jul 13;8(1):10617. Epub 2018 Jul 13.

La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC 3086, Australia.

The jerantinine family of Aspidosperma indole alkaloids from Tabernaemontana corymbosa are potent microtubule-targeting agents with broad spectrum anticancer activity. The natural supply of these precious metabolites has been significantly disrupted due to the inclusion of T. corymbosa on the endangered list of threatened species by the International Union for Conservation of Nature. This report describes the asymmetric syntheses of (-)-jerantinines A and E from sustainably sourced (-)-tabersonine, using a straight-forward and robust biomimetic approach. Biological investigations of synthetic (-)-jerantinine A, along with molecular modelling and X-ray crystallography studies of the tubulin-(-)-jerantinine B acetate complex, advocate an anticancer mode of action of the jerantinines operating via microtubule disruption resulting from binding at the colchicine site. This work lays the foundation for accessing useful quantities of enantiomerically pure jerantinine alkaloids for future development.
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http://dx.doi.org/10.1038/s41598-018-28880-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6045569PMC
July 2018