Publications by authors named "Tarun M Kapoor"

103 Publications

A chemical genetics approach to examine the functions of AAA proteins.

Nat Struct Mol Biol 2021 Apr 29;28(4):388-397. Epub 2021 Mar 29.

Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY, USA.

The structural conservation across the AAA (ATPases associated with diverse cellular activities) protein family makes designing selective chemical inhibitors challenging. Here, we identify a triazolopyridine-based fragment that binds the AAA domain of human katanin, a microtubule-severing protein. We have developed a model for compound binding and designed ASPIR-1 (allele-specific, proximity-induced reactivity-based inhibitor-1), a cell-permeable compound that selectively inhibits katanin with an engineered cysteine mutation. Only in cells expressing mutant katanin does ASPIR-1 treatment increase the accumulation of CAMSAP2 at microtubule minus ends, confirming specific on-target cellular activity. Importantly, ASPIR-1 also selectively inhibits engineered cysteine mutants of human VPS4B and FIGL1-AAA proteins, involved in organelle dynamics and genome stability, respectively. Structural studies confirm our model for compound binding at the AAA ATPase site and the proximity-induced reactivity-based inhibition. Together, our findings suggest a chemical genetics approach to decipher AAA protein functions across essential cellular processes and to test hypotheses for developing therapeutics.
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http://dx.doi.org/10.1038/s41594-021-00575-9DOI Listing
April 2021

Biochemical reconstitutions reveal principles of human γ-TuRC assembly and function.

J Cell Biol 2021 Mar;220(3)

Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY.

The formation of cellular microtubule networks is regulated by the γ-tubulin ring complex (γ-TuRC). This ∼2.3 MD assembly of >31 proteins includes γ-tubulin and GCP2-6, as well as MZT1 and an actin-like protein in a "lumenal bridge" (LB). The challenge of reconstituting the γ-TuRC has limited dissections of its assembly and function. Here, we report a biochemical reconstitution of the human γ-TuRC (γ-TuRC-GFP) as a ∼35 S complex that nucleates microtubules in vitro. In addition, we generate a subcomplex, γ-TuRCΔLB-GFP, which lacks MZT1 and actin. We show that γ-TuRCΔLB-GFP nucleates microtubules in a guanine nucleotide-dependent manner and with similar efficiency as the holocomplex. Electron microscopy reveals that γ-TuRC-GFP resembles the native γ-TuRC architecture, while γ-TuRCΔLB-GFP adopts a partial cone shape presenting only 8-10 γ-tubulin subunits and lacks a well-ordered lumenal bridge. Our results show that the γ-TuRC can be reconstituted using a limited set of proteins and suggest that the LB facilitates the self-assembly of regulatory interfaces around a microtubule-nucleating "core" in the holocomplex.
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http://dx.doi.org/10.1083/jcb.202009146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7844428PMC
March 2021

Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase.

Biophys J 2021 03 17;120(6):1020-1030. Epub 2020 Dec 17.

Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, New York. Electronic address:

The superfamily 1 helicase nonstructural protein 13 (nsp13) is required for SARS-CoV-2 replication. The mechanism and regulation of nsp13 has not been explored at the single-molecule level. Specifically, force-dependent unwinding experiments have yet to be performed for any coronavirus helicase. Here, using optical tweezers, we find that nsp13 unwinding frequency, processivity, and velocity increase substantially when a destabilizing force is applied to the RNA substrate. These results, along with bulk assays, depict nsp13 as an intrinsically weak helicase that can be activated >50-fold by piconewton forces. Such force-dependent behavior contrasts the known behavior of other viral monomeric helicases, such as hepatitis C virus NS3, and instead draws stronger parallels to ring-shaped helicases. Our findings suggest that mechanoregulation, which may be provided by a directly bound RNA-dependent RNA polymerase, enables on-demand helicase activity on the relevant polynucleotide substrate during viral replication.
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http://dx.doi.org/10.1016/j.bpj.2020.11.2276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7837305PMC
March 2021

Microtubules Enhance Mesoscale Effective Diffusivity in the Crowded Metaphase Cytoplasm.

Dev Cell 2020 09 19;54(5):574-582.e4. Epub 2020 Aug 19.

Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY 10065, USA. Electronic address:

Mesoscale macromolecular complexes and organelles, tens to hundreds of nanometers in size, crowd the eukaryotic cytoplasm. It is therefore unclear how mesoscale particles remain sufficiently mobile to regulate dynamic processes such as cell division. Here, we study mobility across dividing cells that contain densely packed, dynamic microtubules, comprising the metaphase spindle. In dividing human cells, we tracked 40 nm genetically encoded multimeric nanoparticles (GEMs), whose sizes are commensurate with the inter-filament spacing in metaphase spindles. Unexpectedly, the effective diffusivity of GEMs was similar inside the dense metaphase spindle and the surrounding cytoplasm. Eliminating microtubules or perturbing their polymerization dynamics decreased diffusivity by ~30%, suggesting that microtubule polymerization enhances random displacements to amplify diffusive-like motion. Our results suggest that microtubules effectively fluidize the mitotic cytoplasm to equalize mesoscale mobility across a densely packed, dynamic, non-uniform environment, thus spatially maintaining a key biophysical parameter that impacts biochemistry, ranging from metabolism to the nucleation of cytoskeletal filaments.
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http://dx.doi.org/10.1016/j.devcel.2020.07.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685229PMC
September 2020

Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex.

Cell 2020 09 28;182(6):1560-1573.e13. Epub 2020 Jul 28.

Laboratory of Molecular Biophysics, The Rockefeller University, New York, NY 10065, USA. Electronic address:

SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated and transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp8/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryoelectron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template product in complex with two molecules of the nsp13 helicase. The Nidovirales order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12 thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapy development.
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http://dx.doi.org/10.1016/j.cell.2020.07.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7386476PMC
September 2020

Purification of Affinity Tag-free Recombinant Tubulin from Insect Cells.

STAR Protoc 2020 Jun 3;1(1). Epub 2020 Jun 3.

Laboratory of Chemistry and Cell Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.

α/β-tubulin heterodimers, which can harbor diverse isotypes and post-translational modifications, polymerize into microtubules that are fundamental to many cellular processes. Due to long-standing challenges in generating recombinant tubulin, however, it has been difficult to examine the properties of specific tubulin isotypes. Here, we provide a protocol for purifying milligrams of affinity tag-free, isotypically pure recombinant tubulin. Our method can be applicable to any tubulin of interest, opening the door to dissecting how tubulin diversity regulates microtubule function. For complete details on the use and execution of this protocol, please see Ti et al. (2018).
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http://dx.doi.org/10.1016/j.xpro.2019.100011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7416841PMC
June 2020

Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase.

bioRxiv 2020 Jul 31. Epub 2020 Jul 31.

The superfamily-1 helicase non-structural protein 13 (nsp13) is required for SARS-CoV-2 replication, making it an important antiviral therapeutic target. The mechanism and regulation of nsp13 has not been explored at the single-molecule level. Specifically, force-dependent unwinding experiments have yet to be performed for any coronavirus helicase. Here, using optical tweezers, we find that nsp13 unwinding frequency, processivity, and velocity increase substantially when a destabilizing force is applied to the dsRNA, suggesting a passive unwinding mechanism. These results, along with bulk assays, depict nsp13 as an intrinsically weak helicase that can be potently activated by picoNewton forces. Such force-dependent behavior contrasts the known behavior of other viral monomeric helicases, drawing stronger parallels to ring-shaped helicases. Our findings suggest that mechanoregulation, which may be provided by a directly bound RNA-dependent RNA polymerase, enables on-demand helicase activity on the relevant polynucleotide substrate during viral replication.
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http://dx.doi.org/10.1101/2020.07.31.231274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402037PMC
July 2020

Chemical strategies to overcome resistance against targeted anticancer therapeutics.

Nat Chem Biol 2020 08 21;16(8):817-825. Epub 2020 Jul 21.

Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY, USA.

Emergence of resistance is a major factor limiting the efficacy of molecularly targeted anticancer drugs. Understanding the specific mutations, or other genetic or cellular changes, that confer drug resistance can help in the development of therapeutic strategies with improved efficacies. Here, we outline recent progress in understanding chemotype-specific mechanisms of resistance and present chemical strategies, such as designing drugs with distinct binding modes or using proteolysis targeting chimeras, to overcome resistance. We also discuss how targeting multiple binding sites with bifunctional inhibitors or identifying collateral sensitivity profiles can be exploited to limit the emergence of resistance. Finally, we highlight how incorporating analyses of resistance early in drug development can help with the design and evaluation of therapeutics that can have long-term benefits for patients.
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http://dx.doi.org/10.1038/s41589-020-0596-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510053PMC
August 2020

Long-range intramolecular allostery and regulation in the dynein-like AAA protein Mdn1.

Proc Natl Acad Sci U S A 2020 08 21;117(31):18459-18469. Epub 2020 Jul 21.

Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY 10065;

Mdn1 is an essential mechanoenzyme that uses the energy from ATP hydrolysis to physically reshape and remodel, and thus mature, the 60S subunit of the ribosome. This massive (>500 kDa) protein has an N-terminal AAA (ATPase associated with diverse cellular activities) ring, which, like dynein, has six ATPase sites. The AAA ring is followed by large (>2,000 aa) linking domains that include an ∼500-aa disordered (D/E-rich) region, and a C-terminal substrate-binding MIDAS domain. Recent models suggest that intramolecular docking of the MIDAS domain onto the AAA ring is required for Mdn1 to transmit force to its ribosomal substrates, but it is not currently understood what role the linking domains play, or why tethering the MIDAS domain to the AAA ring is required for protein function. Here, we use chemical probes, single-particle electron microscopy, and native mass spectrometry to study the AAA and MIDAS domains separately or in combination. We find that Mdn1 lacking the D/E-rich and MIDAS domains retains ATP and chemical probe binding activities. Free MIDAS domain can bind to the AAA ring of this construct in a stereo-specific bimolecular interaction, and, interestingly, this binding reduces ATPase activity. Whereas intramolecular MIDAS docking appears to require a treatment with a chemical inhibitor or preribosome binding, bimolecular MIDAS docking does not. Hence, tethering the MIDAS domain to the AAA ring serves to prevent, rather than promote, MIDAS docking in the absence of inducing signals.
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http://dx.doi.org/10.1073/pnas.2002792117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7414173PMC
August 2020

MZT Proteins Form Multi-Faceted Structural Modules in the γ-Tubulin Ring Complex.

Cell Rep 2020 06;31(13):107791

Laboratory of Chemistry and Cell Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA. Electronic address:

Microtubule organization depends on the γ-tubulin ring complex (γ-TuRC), a ∼2.3-MDa nucleation factor comprising an asymmetric assembly of γ-tubulin and GCP2-GCP6. However, it is currently unclear how the γ-TuRC-associated microproteins MZT1 and MZT2 contribute to the structure and regulation of the holocomplex. Here, we report cryo-EM structures of MZT1 and MZT2 in the context of the native human γ-TuRC. MZT1 forms two subcomplexes with the N-terminal α-helical domains of GCP3 or GCP6 (GCP-NHDs) within the γ-TuRC "lumenal bridge." We determine the X-ray structure of recombinant MZT1/GCP6-NHD and find it is similar to that within the native γ-TuRC. We identify two additional MZT/GCP-NHD-like subcomplexes, one of which is located on the outer face of the γ-TuRC and comprises MZT2 and GCP2-NHD in complex with a centrosomin motif 1 (CM1)-containing peptide. Our data reveal how MZT1 and MZT2 establish multi-faceted, structurally mimetic "modules" that can expand structural and regulatory interfaces in the γ-TuRC.
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http://dx.doi.org/10.1016/j.celrep.2020.107791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7416306PMC
June 2020

Distinct Mechanisms of Resistance to a CENP-E Inhibitor Emerge in Near-Haploid and Diploid Cancer Cells.

Cell Chem Biol 2020 07 21;27(7):850-857.e6. Epub 2020 May 21.

Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY 10065, USA. Electronic address:

Aberrant chromosome numbers in cancer cells may impose distinct constraints on the emergence of drug resistance-a major factor limiting the long-term efficacy of molecularly targeted therapeutics. However, for most anticancer drugs we lack analyses of drug-resistance mechanisms in cells with different karyotypes. Here, we focus on GSK923295, a mitotic kinesin CENP-E inhibitor that was evaluated in clinical trials as a cancer therapeutic. We performed unbiased selections to isolate inhibitor-resistant clones in diploid and near-haploid cancer cell lines. In diploid cells we identified single-point mutations that can suppress inhibitor binding. In contrast,transcriptome analyses revealed that the C-terminus of CENP-E was disrupted in GSK923295-resistant near-haploid cells. While chemical inhibition of CENP-E is toxic to near-haploid cells, knockout of the CENPE gene does not suppress haploid cell proliferation, suggesting that deletion of the CENP-E C-terminus can confer resistance to GSK923295. Together, these findings indicate that different chromosome copy numbers in cells can alter epistatic dependencies and lead to distinct modes of chemotype-specific resistance.
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http://dx.doi.org/10.1016/j.chembiol.2020.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7444662PMC
July 2020

Asymmetric Molecular Architecture of the Human γ-Tubulin Ring Complex.

Cell 2020 01 17;180(1):165-175.e16. Epub 2019 Dec 17.

Laboratory of Chemistry and Cell Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA. Electronic address:

The γ-tubulin ring complex (γ-TuRC) is an essential regulator of centrosomal and acentrosomal microtubule formation, yet its structure is not known. Here, we present a cryo-EM reconstruction of the native human γ-TuRC at ∼3.8 Å resolution, revealing an asymmetric, cone-shaped structure. Pseudo-atomic models indicate that GCP4, GCP5, and GCP6 form distinct Y-shaped assemblies that structurally mimic GCP2/GCP3 subcomplexes distal to the γ-TuRC "seam." We also identify an unanticipated structural bridge that includes an actin-like protein and spans the γ-TuRC lumen. Despite its asymmetric architecture, the γ-TuRC arranges γ-tubulins into a helical geometry poised to nucleate microtubules. Diversity in the γ-TuRC subunits introduces large (>100,000 Å) surfaces in the complex that allow for interactions with different regulatory factors. The observed compositional complexity of the γ-TuRC could self-regulate its assembly into a cone-shaped structure to control microtubule formation across diverse contexts, e.g., within biological condensates or alongside existing filaments.
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http://dx.doi.org/10.1016/j.cell.2019.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7027161PMC
January 2020

Analyzing Resistance to Design Selective Chemical Inhibitors for AAA Proteins.

Cell Chem Biol 2019 09 27;26(9):1263-1273.e5. Epub 2019 Jun 27.

Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY 10065, USA. Electronic address:

Drug-like inhibitors are often designed by mimicking cofactor or substrate interactions with enzymes. However, as active sites are comprised of conserved residues, it is difficult to identify the critical interactions needed to design selective inhibitors. We are developing an approach, named RADD (resistance analysis during design), which involves engineering point mutations in the target to generate active alleles and testing compounds against them. Mutations that alter compound potency identify residues that make key interactions with the inhibitor and predict target-binding poses. Here, we apply this approach to analyze how diaminotriazole-based inhibitors bind spastin, a microtubule-severing AAA (ATPase associated with diverse cellular activities) protein. The distinct binding poses predicted for two similar inhibitors were confirmed by a series of X-ray structures. Importantly, our approach not only reveals how selective inhibition of the target can be achieved but also identifies resistance-conferring mutations at the early stages of the design process.
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http://dx.doi.org/10.1016/j.chembiol.2019.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754270PMC
September 2019

High-resolution imaging reveals how the spindle midzone impacts chromosome movement.

J Cell Biol 2019 08 27;218(8):2529-2544. Epub 2019 Jun 27.

Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY

In the spindle midzone, microtubules from opposite half-spindles form bundles between segregating chromosomes. Microtubule bundles can either push or restrict chromosome movement during anaphase in different cellular contexts, but how these activities are achieved remains poorly understood. Here, we use high-resolution live-cell imaging to analyze individual microtubule bundles, growing filaments, and chromosome movement in dividing human cells. Within bundles, filament overlap length marked by the cross-linking protein PRC1 decreases during anaphase as chromosome segregation slows. Filament ends within microtubule bundles appear capped despite dynamic PRC1 turnover and submicrometer proximity to growing microtubules. Chromosome segregation distance and rate are increased in two human cell lines when microtubule bundle assembly is prevented via PRC1 knockdown. Upon expressing a mutant PRC1 with reduced microtubule affinity, bundles assemble but chromosome hypersegregation is still observed. We propose that microtubule overlap length reduction, typically linked to pushing forces generated within filament bundles, is needed to properly restrict spindle elongation and position chromosomes within daughter cells.
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http://dx.doi.org/10.1083/jcb.201904169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6683753PMC
August 2019

Using chemical inhibitors to probe AAA protein conformational dynamics and cellular functions.

Curr Opin Chem Biol 2019 06 23;50:45-54. Epub 2019 Mar 23.

Laboratory of Chemistry and Cell Biology, Rockefeller University, New York, United States. Electronic address:

The AAA proteins are a family of enzymes that play key roles in diverse dynamic cellular processes, ranging from proteostasis to directional intracellular transport. Dysregulation of AAA proteins has been linked to several diseases, including cancer, suggesting a possible therapeutic role for inhibitors of these enzymes. In the past decade, new chemical probes have been developed for AAA proteins including p97, dynein, midasin, and ClpC1. In this review, we discuss how these compounds have been used to study the cellular functions and conformational dynamics of AAA proteins. We discuss future directions for inhibitor development and early efforts to utilize AAA protein inhibitors in the clinical setting.
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http://dx.doi.org/10.1016/j.cbpa.2019.02.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6880239PMC
June 2019

Designing Allele-Specific Inhibitors of Spastin, a Microtubule-Severing AAA Protein.

J Am Chem Soc 2019 04 27;141(14):5602-5606. Epub 2019 Mar 27.

Laboratory of Chemistry and Cell Biology , The Rockefeller University , New York , New York 10065 , United States.

The bump-hole approach is a powerful chemical biology strategy to specifically probe the functions of closely related proteins. However, for many protein families, such as the ATPases associated with diverse cellular activities (AAA), we lack structural data for inhibitor-protein complexes to design allele-specific chemical probes. Here we report the X-ray structure of a pyrazolylaminoquinazoline-based inhibitor bound to spastin, a microtubule-severing AAA protein, and characterize the residues involved in inhibitor binding. We show that an inhibitor analogue with a single-atom hydrogen-to-fluorine modification can selectively target a spastin allele with an engineered cysteine mutation in its active site. We also report an X-ray structure of the fluoro analogue bound to the spastin mutant. Furthermore, analyses of other mutant alleles suggest how the stereoelectronics of the fluorine-cysteine interaction, rather than sterics alone, contribute to the inhibitor-allele selectivity. This approach could be used to design allele-specific probes for studying cellular functions of spastin isoforms. Our data also suggest how tuning stereoelectronics can lead to specific inhibitor-allele pairs for the AAA superfamily.
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http://dx.doi.org/10.1021/jacs.8b13257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6637947PMC
April 2019

Designing a chemical inhibitor for the AAA protein spastin using active site mutations.

Nat Chem Biol 2019 05 18;15(5):444-452. Epub 2019 Feb 18.

Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY, USA.

Spastin is a microtubule-severing AAA (ATPases associated with diverse cellular activities) protein needed for cell division and intracellular vesicle transport. Currently, we lack chemical inhibitors to probe spastin function in such dynamic cellular processes. To design a chemical inhibitor of spastin, we tested selected heterocyclic scaffolds against wild-type protein and constructs with engineered mutations in the nucleotide-binding site that do not substantially disrupt ATPase activity. These data, along with computational docking, guided improvements in compound potency and selectivity and led to spastazoline, a pyrazolyl-pyrrolopyrimidine-based cell-permeable probe for spastin. These studies also identified spastazoline-resistance-conferring point mutations in spastin. Spastazoline, along with the matched inhibitor-sensitive and inhibitor-resistant cell lines we generated, were used in parallel experiments to dissect spastin-specific phenotypes in dividing cells. Together, our findings suggest how chemical probes for AAA proteins, along with inhibitor resistance-conferring mutations, can be designed and used to dissect dynamic cellular processes.
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http://dx.doi.org/10.1038/s41589-019-0225-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6558985PMC
May 2019

Structural Insights into Mdn1, an Essential AAA Protein Required for Ribosome Biogenesis.

Cell 2018 10 11;175(3):822-834.e18. Epub 2018 Oct 11.

Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY 10065, USA. Electronic address:

Mdn1 is an essential AAA (ATPase associated with various activities) protein that removes assembly factors from distinct precursors of the ribosomal 60S subunit. However, Mdn1's large size (∼5,000 amino acid [aa]) and its limited homology to other well-studied proteins have restricted our understanding of its remodeling function. Here, we present structures for S. pombe Mdn1 in the presence of AMPPNP at up to ∼4 Å or ATP plus Rbin-1, a chemical inhibitor, at ∼8 Å resolution. These data reveal that Mdn1's MIDAS domain is tethered to its ring-shaped AAA domain through an ∼20 nm long structured linker and a flexible ∼500 aa Asp/Glu-rich motif. We find that the MIDAS domain, which also binds other ribosome-assembly factors, docks onto the AAA ring in a nucleotide state-specific manner. Together, our findings reveal how conformational changes in the AAA ring can be directly transmitted to the MIDAS domain and thereby drive the targeted release of assembly factors from ribosomal 60S-subunit precursors.
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http://dx.doi.org/10.1016/j.cell.2018.09.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6289053PMC
October 2018

Human β-Tubulin Isotypes Can Regulate Microtubule Protofilament Number and Stability.

Dev Cell 2018 10 20;47(2):175-190.e5. Epub 2018 Sep 20.

Laboratory of Chemistry and Cell Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA. Electronic address:

Cell biological studies have shown that protofilament number, a fundamental feature of microtubules, can correlate with the expression of different tubulin isotypes. However, it is not known if tubulin isotypes directly control this basic microtubule property. Here, we report high-resolution cryo-EM reconstructions (3.5-3.65 Å) of purified human α1B/β3 and α1B/β2B microtubules and find that the β-tubulin isotype can determine protofilament number. Comparisons of atomic models of 13- and 14-protofilament microtubules reveal how tubulin subunit plasticity, manifested in "accordion-like" distributed structural changes, can accommodate distinct lattice organizations. Furthermore, compared to α1B/β3 microtubules, α1B/β2B filaments are more stable to passive disassembly and against depolymerization by MCAK or chTOG, microtubule-associated proteins with distinct mechanisms of action. Mixing tubulin isotypes in different proportions results in microtubules with protofilament numbers and stabilities intermediate to those of isotypically pure filaments. Together, our findings indicate that microtubule protofilament number and stability can be controlled through β-tubulin isotype composition.
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http://dx.doi.org/10.1016/j.devcel.2018.08.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6362463PMC
October 2018

Analyzing the micromechanics of the cell division apparatus.

Methods Cell Biol 2018 1;145:173-190. Epub 2018 May 1.

Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY, United States. Electronic address:

Cell division involves mechanical processes, such as chromosome transport and centrosome separation. Quantitative micromanipulation-based approaches have been central to dissecting the forces driving these processes. We highlight two biophysical assays that can be employed for such analyses. First, an in vitro "mini-spindle" assay is described that can be used to examine the collective mechanics of mitotic motor proteins cross-linking two microtubules. In the spindle, motor proteins (e.g., kinesin-5, kinesin-14, and dynein) can localize to overlapping microtubules that slide relative to each other, work as an ensemble, and equilibrate between cytoplasm and the microtubules. The "mini-spindle" assay can recapitulate these features and allows measurements of forces generated between adjacent microtubules and their dependence on filament orientation, sliding speed, overlap length, and motor protein density. Second, we describe a force-calibrated microneedle-based "whole-spindle" micromechanics assay. Microneedle-based micromanipulation can be a useful technique to examine cellular scale mechanics, but its use has been restricted by the difficulty in getting probes to penetrate the plasma membrane without disrupting cell physiology. As detailed here, the use of cell-free extracts prepared from metaphase-arrested Xenopus eggs can address this limitation. These micromanipulation studies also benefit from the use of frozen stocks of Xenopus egg extract. Together, these approaches can be used to decipher how micromechanics and biochemical activities ensure successful cell division.
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http://dx.doi.org/10.1016/bs.mcb.2018.03.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6029715PMC
December 2018

A Chemical Proteomics Approach to Reveal Direct Protein-Protein Interactions in Living Cells.

Cell Chem Biol 2018 01 5;25(1):110-120.e3. Epub 2017 Nov 5.

Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY 10065, USA. Electronic address:

Protein-protein interactions mediate essential cellular processes, however the detection of native interactions is challenging since they are often low affinity and context dependent. Here, we develop a chemical proteomics approach in vivo CLASPI [iCLASPI] (in vivo crosslinking-assisted and stable isotope labeling by amino acids in cell culture [SILAC]-based protein identification) relying upon photo-crosslinking, amber suppression, and SILAC-based quantitative proteomics to profile context-dependent protein-protein interactions in living cells. First, we use iCLASPI to profile in vivo binding partners of the N-terminal tails of soluble histone H3 or H4. We identify known histone chaperones and modifying proteins, thereby validating our approach, and find an interaction between soluble histone H3 and UBR7, an E3 ubiquitin ligase, mediated by UBR7's PHD domain. Furthermore, we apply iCLASPI to profile the context-dependent protein-protein interactions of chromatin-associated histone H3 at different cell-cycle stages, and identify ANP32A as a mitosis-specific interactor. Our results demonstrate that the iCLASPI approach can provide a general strategy for identifying native, context-dependent direct protein-protein interactions using photo-crosslinking and quantitative proteomics.
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http://dx.doi.org/10.1016/j.chembiol.2017.10.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5775914PMC
January 2018

Leveraging Chemotype-Specific Resistance for Drug Target Identification and Chemical Biology.

Trends Pharmacol Sci 2017 12 13;38(12):1100-1109. Epub 2017 Oct 13.

Laboratory of Chemistry and Cell Biology, The Rockefeller University, 1200 York Ave., New York, NY 10065, USA.

Identifying the direct physiological targets of drugs and chemical probes remains challenging. Here we describe how resistance can be used to achieve 'gold-standard' validation of a chemical inhibitor's direct target in human cells. This involves demonstrating that a silent mutation in the target that suppresses inhibitor activity in cell-based assays can also reduce inhibitor potency in biochemical assays. Further, phenotypes due to target inhibition can be identified as those observed in the inhibitor-sensitive cells, across a range of inhibitor concentrations, but not in genetically matched cells with a silent resistance-conferring mutation in the target. We propose that chemotype-specific resistance, which is generally considered to be a limitation of molecularly targeted agents, can be leveraged to deconvolve the mechanism of action of drugs and to properly use chemical probes.
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http://dx.doi.org/10.1016/j.tips.2017.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5708298PMC
December 2017

Chemical structure-guided design of dynapyrazoles, cell-permeable dynein inhibitors with a unique mode of action.

Elife 2017 05 19;6. Epub 2017 May 19.

Laboratory of Chemistry and Cell Biology, Rockefeller University, New York, United States.

Cytoplasmic dyneins are motor proteins in the AAA+ superfamily that transport cellular cargos toward microtubule minus-ends. Recently, ciliobrevins were reported as selective cell-permeable inhibitors of cytoplasmic dyneins. As is often true for first-in-class inhibitors, the use of ciliobrevins has in part been limited by low potency. Moreover, suboptimal chemical properties, such as the potential to isomerize, have hindered efforts to improve ciliobrevins. Here, we characterized the structure of ciliobrevins and designed conformationally constrained isosteres. These studies identified dynapyrazoles, inhibitors more potent than ciliobrevins. At single-digit micromolar concentrations dynapyrazoles block intraflagellar transport in the cilium and lysosome motility in the cytoplasm, processes that depend on cytoplasmic dyneins. Further, we find that while ciliobrevins inhibit both dynein's microtubule-stimulated and basal ATPase activity, dynapyrazoles strongly block only microtubule-stimulated activity. Together, our studies suggest that chemical-structure-based analyses can lead to inhibitors with improved properties and distinct modes of inhibition.
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http://dx.doi.org/10.7554/eLife.25174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5478271PMC
May 2017

The mechanics of microtubule networks in cell division.

J Cell Biol 2017 06 10;216(6):1525-1531. Epub 2017 May 10.

Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY 10065

The primary goal of a dividing somatic cell is to accurately and equally segregate its genome into two new daughter cells. In eukaryotes, this process is performed by a self-organized structure called the mitotic spindle. It has long been appreciated that mechanical forces must be applied to chromosomes. At the same time, the network of microtubules in the spindle must be able to apply and sustain large forces to maintain spindle integrity. Here we consider recent efforts to measure forces generated within microtubule networks by ensembles of key proteins. New findings, such as length-dependent force generation, protein clustering by asymmetric friction, and entropic expansion forces will help advance models of force generation needed for spindle function and maintaining integrity.
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http://dx.doi.org/10.1083/jcb.201612064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5461028PMC
June 2017

Metaphase Spindle Assembly.

Authors:
Tarun M Kapoor

Biology (Basel) 2017 Feb 3;6(1). Epub 2017 Feb 3.

Laboratory of Chemistry and Cell Biology, the Rockefeller University, New York, NY 10065, USA.

A microtubule-based bipolar spindle is required for error-free chromosome segregation during cell division. In this review I discuss the molecular mechanisms required for the assembly of this dynamic micrometer-scale structure in animal cells.
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http://dx.doi.org/10.3390/biology6010008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5372001PMC
February 2017

Potent, Reversible, and Specific Chemical Inhibitors of Eukaryotic Ribosome Biogenesis.

Cell 2016 Oct 22;167(2):512-524.e14. Epub 2016 Sep 22.

Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY 10065, USA. Electronic address:

All cellular proteins are synthesized by ribosomes, whose biogenesis in eukaryotes is a complex multi-step process completed within minutes. Several chemical inhibitors of ribosome function are available and used as tools or drugs. By contrast, we lack potent validated chemical probes to analyze the dynamics of eukaryotic ribosome assembly. Here, we combine chemical and genetic approaches to discover ribozinoindoles (or Rbins), potent and reversible triazinoindole-based inhibitors of eukaryotic ribosome biogenesis. Analyses of Rbin sensitivity and resistance conferring mutations in fission yeast, along with biochemical assays with recombinant proteins, provide evidence that Rbins' physiological target is Midasin, an essential ∼540-kDa AAA+ (ATPases associated with diverse cellular activities) protein. Using Rbins to acutely inhibit or activate Midasin function, in parallel experiments with inhibitor-sensitive or inhibitor-resistant cells, we uncover Midasin's role in assembling Nsa1 particles, nucleolar precursors of the 60S subunit. Together, our findings demonstrate that Rbins are powerful probes for eukaryotic ribosome assembly.
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http://dx.doi.org/10.1016/j.cell.2016.08.070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5116814PMC
October 2016

Near-atomic cryo-EM structure of PRC1 bound to the microtubule.

Proc Natl Acad Sci U S A 2016 08 4;113(34):9430-9. Epub 2016 Aug 4.

Molecular Biophysics and Integrative Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; Howard Hughes Medical Institute, University of California, Berkeley, CA 94720; California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720; Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720

Proteins that associate with microtubules (MTs) are crucial to generate MT arrays and establish different cellular architectures. One example is PRC1 (protein regulator of cytokinesis 1), which cross-links antiparallel MTs and is essential for the completion of mitosis and cytokinesis. Here we describe a 4-Å-resolution cryo-EM structure of monomeric PRC1 bound to MTs. Residues in the spectrin domain of PRC1 contacting the MT are highly conserved and interact with the same pocket recognized by kinesin. We additionally found that PRC1 promotes MT assembly even in the presence of the MT stabilizer taxol. Interestingly, the angle of the spectrin domain on the MT surface corresponds to the previously observed cross-bridge angle between MTs cross-linked by full-length, dimeric PRC1. This finding, together with molecular dynamic simulations describing the intrinsic flexibility of PRC1, suggests that the MT-spectrin domain interface determines the geometry of the MT arrays cross-linked by PRC1.
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http://dx.doi.org/10.1073/pnas.1609903113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5003279PMC
August 2016

The structured core of human β tubulin confers isotype-specific polymerization properties.

J Cell Biol 2016 05 16;213(4):425-33. Epub 2016 May 16.

Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY 10065

Diversity in cytoskeleton organization and function may be achieved through variations in primary sequence of tubulin isotypes. Recently, isotype functional diversity has been linked to a "tubulin code" in which the C-terminal tail, a region of substantial sequence divergence between isotypes, specifies interactions with microtubule-associated proteins. However, it is not known whether residue changes in this region alter microtubule dynamic instability. Here, we examine recombinant tubulin with human β isotype IIB and characterize polymerization dynamics. Microtubules with βIIB have catastrophe frequencies approximately threefold lower than those with isotype βIII, a suppression similar to that achieved by regulatory proteins. Further, we generate chimeric β tubulins with native tail sequences swapped between isotypes. These chimeras have catastrophe frequencies similar to that of the corresponding full-length construct with the same core sequence. Together, our data indicate that residue changes within the conserved β tubulin core are largely responsible for the observed isotype-specific changes in dynamic instability parameters and tune tubulin's polymerization properties across a wide range.
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http://dx.doi.org/10.1083/jcb.201603050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4878094PMC
May 2016

Mutations in Human Tubulin Proximal to the Kinesin-Binding Site Alter Dynamic Instability at Microtubule Plus- and Minus-Ends.

Dev Cell 2016 Apr;37(1):72-84

Laboratory of Chemistry and Cell Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA. Electronic address:

The assembly of microtubule-based cellular structures depends on regulated tubulin polymerization and directional transport. Here, we purify and characterize tubulin heterodimers that have human β-tubulin isotype III (TUBB3), as well as heterodimers with one of two β-tubulin mutations (D417H or R262H). Both point mutations are proximal to the kinesin-binding site and have been linked to an ocular motility disorder in humans. Compared to wild-type, microtubules with these mutations have decreased catastrophe frequencies and increased average lifetimes of plus- and minus-end-stabilizing caps. Importantly, the D417H mutation does not alter microtubule lattice structure or Mal3 binding to growing filaments. Instead, this mutation reduces the affinity of tubulin for TOG domains and colchicine, suggesting that the distribution of tubulin heterodimer conformations is changed. Together, our findings reveal how residues on the surface of microtubules, distal from the GTP-hydrolysis site and inter-subunit contacts, can alter polymerization dynamics at the plus- and minus-ends of microtubules.
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http://dx.doi.org/10.1016/j.devcel.2016.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4832424PMC
April 2016

Cytoplasmic Dynein Antagonists with Improved Potency and Isoform Selectivity.

ACS Chem Biol 2016 Jan 11;11(1):53-60. Epub 2015 Nov 11.

Laboratory of Chemistry and Cell Biology, Rockefeller University , New York City, New York 10065, United States.

Cytoplasmic dyneins 1 and 2 are related members of the AAA+ superfamily (ATPases associated with diverse cellular activities) that function as the predominant minus-end-directed microtubule motors in eukaryotic cells. Dynein 1 controls mitotic spindle assembly, organelle movement, axonal transport, and other cytosolic, microtubule-guided processes, whereas dynein 2 mediates retrograde trafficking within motile and primary cilia. Small-molecule inhibitors are important tools for investigating motor protein-dependent mechanisms, and ciliobrevins were recently discovered as the first dynein-specific chemical antagonists. Here, we demonstrate that ciliobrevins directly target the heavy chains of both dynein isoforms and explore the structure-activity landscape of these inhibitors in vitro and in cells. In addition to identifying chemical motifs that are essential for dynein blockade, we have discovered analogs with increased potency and dynein 2 selectivity. These antagonists effectively disrupt Hedgehog signaling, intraflagellar transport, and ciliogenesis, making them useful probes of these and other cytoplasmic dynein 2-dependent cellular processes.
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http://dx.doi.org/10.1021/acschembio.5b00895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4715766PMC
January 2016