Structure of the γ-tubulin ring complex-capped microtubule.

Microtubules are composed of α-tubulin and ß-tubulin dimers positioned head-to-tail to form protofilaments that associate laterally in varying numbers. It is not known how cellular microtubules assemble with the canonical 13-protofilament architecture, resulting in micrometer-scale α/ß-tubulin tracks for intracellular transport that align with, rather than spiral along, the long axis of the filament. We report that the human ~2.3 MDa γ-tubulin ring complex (γ-TuRC), an essential regulator of microtubule formation that contains 14 γ-tubulins, selectively nucleates 13-protofilament microtubules. Cryogenic electron microscopy reconstructions of γ-TuRC-capped microtubule minus ends reveal the extensive intra-domain and inter-domain motions of γ-TuRC subunits that accommodate luminal bridge components and establish lateral and longitudinal interactions between γ-tubulins and α-tubulins. Our structures suggest that γ-TuRC, an inefficient nucleation template owing to its splayed conformation, can transform into a compacted cap at the microtubule minus end and set the lattice architecture of cellular microtubules.

Spastin regulates anaphase chromosome separation distance and microtubule-containing nuclear tunnels.

Nuclear envelope reassembly during the final stages of each mitosis depends on disassembling spindle microtubules without disrupting chromosome separation. This process involves the transient recruitment of the ESCRT-III complex and spastin, a microtubule-severing AAA (ATPases associated with diverse cellular activities) mechanoenzyme, to late-anaphase chromosomes. However, dissecting mechanisms underlying these rapid processes, which can be completed within minutes, has been difficult. Here, we combine fast-acting chemical inhibitors with live-cell imaging and find that spindle microtubules, along with spastin activity, regulate the number and lifetimes of spastin foci at anaphase chromosomes. Unexpectedly, spastin inhibition impedes chromosome separation, but does not alter the anaphase localization dynamics of CHMP4B, an ESCRT-III protein, or increase γ-H2AX foci, a DNA damage marker. We show spastin inhibition increases the frequency of lamin-lined nuclear microtunnels that can include microtubules penetrating the nucleus. Our findings suggest failure to sever spindle microtubules impedes chromosome separation, yet reforming nuclear envelopes can topologically accommodate persistent microtubules ensuring nuclear DNA is not damaged or exposed to cytoplasm.

Using a Function-First "Scout Fragment"-Based Approach to Develop Allosteric Covalent Inhibitors of Conformationally Dynamic Helicase Mechanoenzymes.

Helicases, classified into six superfamilies, are mechanoenzymes that utilize energy derived from ATP hydrolysis to remodel DNA and RNA substrates. These enzymes have key roles in diverse cellular processes, such as translation, ribosome assembly, and genome maintenance. Helicases with essential functions in certain cancer cells have been identified, and helicases expressed by many viruses are required for their pathogenicity. Therefore, helicases are important targets for chemical probes and therapeutics. However, it has been very challenging to develop chemical inhibitors for helicases, enzymes with high conformational dynamics. We envisioned that electrophilic "scout fragments", which have been used in chemical proteomic studies, could be leveraged to develop covalent inhibitors of helicases. We adopted a function-first approach, combining enzymatic assays with enantiomeric probe pairs and mass spectrometry, to develop a covalent inhibitor that selectively targets an allosteric site in SARS-CoV-2 nsp13, a superfamily-1 helicase. Further, we demonstrate that scout fragments inhibit the activity of two human superfamily-2 helicases, BLM and WRN, involved in genome maintenance. Together, our findings suggest an approach to discover covalent inhibitor starting points and druggable allosteric sites in conformationally dynamic mechanoenzymes.

Structure of the γ-tubulin ring complex-capped microtubule.

Microtubules are composed of α/ß-tubulin dimers positioned head-to-tail to form protofilaments that associate laterally in varying numbers. It is not known how cellular microtubules assemble with the canonical 13-protofilament architecture, resulting in micrometer-scale α/ß-tubulin tracks for intracellular transport that align with, rather than spiral along, the filament's long-axis. We report that the human ∼2.3MDa γ-tubulin ring complex (γ-TuRC), an essential regulator of microtubule formation that contains 14 γ-tubulins, selectively nucleates 13-protofilament microtubules. Cryo-EM reconstructions of γ-TuRC-capped microtubule minus-ends reveal the extensive intra- and inter-domain motions of γ-TuRC subunits that accommodate its actin-containing luminal bridge and establish lateral and longitudinal interactions between γ- and α-tubulins. Our structures reveal how free γ-TuRC, an inefficient nucleation template due to its splayed conformation, transforms into a stable cap that blocks addition or loss of α/ß-tubulins from minus-ends and sets the lattice architecture of cellular microtubules. One Sentence Summary: Structural insights into how the γ-tubulin ring complex nucleates and caps a 13-protofilament microtubule.

Fine-tuning chemical genetics to identify physiologic drug targets.

Genetics-based approaches can enable drug target identification in human cells. In this issue of Cell Chemical Biology, Nguyen et al.1 use inducible degradation of a mismatch repair protein to tune the mutation rate in HCT116 cells, thereby increasing sensitivity and selectivity in identifying resistance-conferring mutations for several cytotoxic small molecules.

Using a function-first 'scout fragment'-based approach to develop allosteric covalent inhibitors of conformationally dynamic helicase mechanoenzymes.

Helicases, classified into six superfamilies, are mechanoenzymes that utilize energy derived from ATP hydrolysis to remodel DNA and RNA substrates. These enzymes have key roles in diverse cellular processes, such as genome replication and maintenance, ribosome assembly and translation. Helicases with essential functions only in certain cancer cells have been identified and helicases expressed by certain viruses are required for their pathogenicity. As a result, helicases are important targets for chemical probes and therapeutics. However, it has been very challenging to develop selective chemical inhibitors for helicases, enzymes with highly dynamic conformations. We envisioned that electrophilic 'scout fragments', which have been used for chemical proteomic based profiling, could be leveraged to develop covalent inhibitors of helicases. We adopted a function-first approach, combining enzymatic assays with enantiomeric probe pairs and mass spectrometry, to develop a covalent inhibitor that selectively targets an allosteric site in SARS-CoV-2 nsp13, a superfamily-1 helicase. Further, we demonstrate that scout fragments inhibit the activity of two human superfamily-2 helicases, BLM and WRN, involved in genome maintenance. Together, our findings suggest a covalent inhibitor discovery approach to target helicases and potentially other conformationally dynamic mechanoenzymes.

High-content microscopy reveals a morphological signature of bortezomib resistance.

Drug resistance is a challenge in anticancer therapy. In many cases, cancers can be resistant to the drug prior to exposure, that is, possess intrinsic drug resistance. However, we lack target-independent methods to anticipate resistance in cancer cell lines or characterize intrinsic drug resistance without a priori knowledge of its cause. We hypothesized that cell morphology could provide an unbiased readout of drug resistance. To test this hypothesis, we used HCT116 cells, a mismatch repair-deficient cancer cell line, to isolate clones that were resistant or sensitive to bortezomib, a well-characterized proteasome inhibitor and anticancer drug to which many cancer cells possess intrinsic resistance. We then expanded these clones and measured high-dimensional single-cell morphology profiles using Cell Painting, a high-content microscopy assay. Our imaging- and computation-based profiling pipeline identified morphological features that differed between resistant and sensitive cells. We used these features to generate a morphological signature of bortezomib resistance. We then employed this morphological signature to analyze a set of HCT116 clones (five resistant and five sensitive) that had not been included in the signature training dataset, and correctly predicted sensitivity to bortezomib in seven cases, in the absence of drug treatment. This signature predicted bortezomib resistance better than resistance to other drugs targeting the ubiquitin-proteasome system, indicating specificity for mechanisms of resistance to bortezomib. Our results establish a proof-of-concept framework for the unbiased analysis of drug resistance using high-content microscopy of cancer cells, in the absence of drug treatment.

Observing inhibition of the SARS-CoV-2 helicase at single-nucleotide resolution.

The genome of SARS-CoV-2 encodes for a helicase (nsp13) that is essential for viral replication and highly conserved across related viruses, making it an attractive antiviral target. Here we use nanopore tweezers, a high-resolution single-molecule technique, to gain detailed insight into how nsp13 turns ATP-hydrolysis into directed motion along nucleic acid strands. We measured nsp13 both as it translocates along single-stranded DNA or unwinds double-stranded DNA. Our data reveal nsp13's single-nucleotide steps, translocating at ∼1000 nt/s or unwinding at ∼100 bp/s. Nanopore tweezers' high spatiotemporal resolution enables detailed kinetic analysis of nsp13 motion. As a proof-of-principle for inhibition studies, we observed nsp13's motion in the presence of the ATPase inhibitor ATPγS. We construct a detailed picture of inhibition in which ATPγS has multiple mechanisms of inhibition. The dominant mechanism of inhibition depends on the application of assisting force. This lays the groundwork for future single-molecule inhibition studies with viral helicases.

Achieving the promise and avoiding the peril of chemical probes using genetics.

Chemical probes can be valuable tools for studying protein targets, but addressing concerns about a probe's cellular target or its specificity can be challenging. A reliable strategy is to use a mutation that does not alter a target's function but confers resistance (or sensitizes) to the inhibitor in both cellular and biochemical assays. However, challenges remain in finding such mutations. Here, we discuss structure- and cell-based approaches to identify resistance- and sensitivity-conferring mutations. Further, we describe how resistance-conferring mutations can help with compound design, and the use of saturation mutagenesis to characterize a compound binding site. We highlight how genetic approaches can ensure the proper use of chemical inhibitors to pursue mechanistic studies and test therapeutic hypotheses.

A nucleotide binding-independent role for γ-tubulin in microtubule capping and cell division.

The γ-tubulin ring complex (γ-TuRC) has essential roles in centrosomal and non-centrosomal microtubule organization during vertebrate mitosis. While there have been important advances in understanding γ-TuRC-dependent microtubule nucleation, γ-TuRC capping of microtubule minus-ends remains poorly characterized. Here, we utilized biochemical reconstitutions and cellular assays to characterize the human γ-TuRC's capping activity. Single filament assays showed that the γ-TuRC remained associated with a nucleated microtubule for tens of minutes. In contrast, caps at dynamic microtubule minus-ends displayed lifetimes of ∼1 min. Reconstituted γ-TuRCs with nucleotide-binding deficient γ-tubulin (γ-tubulinΔGTP) formed ring-shaped complexes that did not nucleate microtubules but capped microtubule minus-ends with lifetimes similar to those measured for wild-type complexes. In dividing cells, microtubule regrowth assays revealed that while knockdown of γ-tubulin suppressed non-centrosomal microtubule formation, add-back of γ-tubulinΔGTP could substantially restore this process. Our results suggest that γ-TuRC capping is a nucleotide-binding-independent activity that plays a role in non-centrosomal microtubule organization during cell division.

Inhibition of the SARS-CoV-2 helicase at single-nucleotide resolution.

The genome of SARS-CoV-2 encodes for a helicase called nsp13 that is essential for viral replication and highly conserved across related viruses, making it an attractive antiviral target. Here we use nanopore tweezers, a high-resolution single-molecule technique, to gain detailed insight into how nsp13 turns ATP-hydrolysis into directed motion along nucleic acid strands. We measured nsp13 both as it translocates along single-stranded DNA or unwinds short DNA duplexes. Our data confirm that nsp13 uses the inchworm mechanism to move along the DNA in single-nucleotide steps, translocating at ~1000 nt/s or unwinding at ~100 bp/s. Nanopore tweezers' high spatio-temporal resolution enables observation of the fundamental physical steps taken by nsp13 even as it translocates at speeds in excess of 1000 nucleotides per second enabling detailed kinetic analysis of nsp13 motion. As a proof-of-principle for inhibition studies, we observed nsp13's motion in the presence of the ATPase inhibitor ATPγS. Our data reveals that ATPγS interferes with nsp13's action by affecting several different kinetic processes. The dominant mechanism of inhibition differs depending on the application of assisting force. These advances demonstrate that nanopore tweezers are a powerful method for studying viral helicase mechanism and inhibition.

Coupling of microtubule bundles isolates them from local disruptions to set the structural stability of the anaphase spindle.

Chromosome segregation requires load-bearing interactions across kinetochore fibers and antiparallel microtubule bundles, which constitute the spindle midzone. Mechanical properties of kinetochore fibers have been characterized during metaphase, when the mitotic spindle achieves steady state. However, it has been difficult to probe the mechanics of the spindle midzone that elongates during anaphase. Here, we combine superresolution expansion and electron microscopies, lattice light-sheet imaging, and laser microsurgery to examine how midzone organization sets its mechanics. We find that individual midzone bundles extend out to multiple positions across chromosomes and form multiple apparent microtubule-based connections with each other. Across the spindle's short axis, these microtubule bundles exhibit restricted, submicrometer-amplitude motions, which are weakly correlated on <10s timescales. Severing individual midzone bundles near their center does not substantially affect positions of neighboring bundles, nor the overall structural stability of the midzone. In contrast, severing multiple midzone bundles or individual bundles at their chromosome-proximal ends significantly displaces neighboring microtubule bundles. Together, these data suggest a model wherein multiple midzone connections both reinforce its structure and mechanically isolate individual bundles from local perturbations. This feature sets the robust midzone architecture to accommodate disruptions, including those which result from lagging chromosomes, and achieve stereotypic outputs, such as proper chromosome separation.

Non-centrosomal microtubules at kinetochores promote rapid chromosome biorientation during mitosis in human cells.

Proper segregation of chromosomes during mitosis depends on "amphitelic attachments"-load-bearing connections of sister kinetochores to the opposite spindle poles via bundles of microtubules, termed as the "K-fibers." Current models of spindle assembly assume that K-fibers arise largely from stochastic capture of microtubules, which occurs at random times and locations and independently at sister kinetochores. We test this assumption by following the movements of all kinetochores in human cells and determine that most amphitelic attachments form synchronously at a specific stage of spindle assembly and within a spatially distinct domain. This biorientation domain is enriched in bundles of antiparallel microtubules, and perturbation of microtubule bundling changes the temporal and spatial dynamics of amphitelic attachment formation. Structural analyses indicate that interactions of kinetochores with microtubule bundles are mediated by non-centrosomal short microtubules that emanate from most kinetochores during early prometaphase. Computational analyses suggest that momentous molecular motor-driven interactions with antiparallel bundles rapidly convert these short microtubules into nascent K-fibers. Thus, load-bearing connections to the opposite spindle poles form simultaneously on sister kinetochores. In contrast to the uncoordinated sequential attachments of sister kinetochores expected in stochastic models of spindle assembly, our model envisions the formation of amphitelic attachments as a deterministic process in which the chromosomes connect with the spindle poles synchronously at a specific stage of spindle assembly and at a defined location determined by the spindle architecture. Experimental analyses of changes in the kinetochore behavior in cells with perturbed activity of molecular motors CenpE and dynein confirm the predictive power of the model.

The MIDAS domain of AAA mechanoenzyme Mdn1 forms catch bonds with two different substrates.

Catch bonds are a form of mechanoregulation wherein protein-ligand interactions are strengthened by the application of dissociative tension. Currently, the best-characterized examples of catch bonds are between single protein-ligand pairs. The essential AAA (ATPase associated with diverse cellular activities) mechanoenzyme Mdn1 drives at least two separate steps in ribosome biogenesis, using its MIDAS domain to extract the ubiquitin-like (UBL) domain-containing proteins Rsa4 and Ytm1 from ribosomal precursors. However, it must subsequently release these assembly factors to reinitiate the enzymatic cycle. The mechanism underlying the switching of the MIDAS-UBL interaction between strongly and weakly bound states is unknown. Here, we use optical tweezers to investigate the force dependence of MIDAS-UBL binding. Parallel experiments with Rsa4 and Ytm1 show that forces up to ~4 pN, matching the magnitude of force produced by AAA proteins similar to Mdn1, enhance the MIDAS domain binding lifetime up to 10-fold, and higher forces accelerate dissociation. Together, our studies indicate that Mdn1's MIDAS domain can form catch bonds with more than one UBL substrate, and provide insights into how mechanoregulation may contribute to the Mdn1 enzymatic cycle during ribosome biogenesis.

Targeting allostery in the Dynein motor domain with small molecule inhibitors.

Cytoplasmic dyneins are AAA (ATPase associated with diverse cellular activities) motor proteins responsible for microtubule minus-end-directed intracellular transport. Dynein's unusually large size, four distinct nucleotide-binding sites, and conformational dynamics pose challenges for the design of potent and selective chemical inhibitors. Here we use structural approaches to develop a model for the inhibition of a well-characterized S. cerevisiae dynein construct by pyrazolo-pyrimidinone-based compounds. These data, along with functional assays of dynein motility and mutagenesis studies, suggest that the compounds inhibit dynein by engaging the regulatory ATPase sites in the AAA3 and AAA4 domains, and not by interacting with dynein's main catalytic site in the AAA1 domain. A double Walker B mutation of the AAA3 and AAA4 sites substantially reduces enzyme activity, suggesting that targeting these regulatory domains is sufficient to inhibit dynein. Our findings reveal how chemical inhibitors can be designed to disrupt allosteric communication across dynein's AAA domains.

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

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.

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

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.

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

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.

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

α/ß-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).