Targeting oncogenic KRasG13C with nucleotide-based covalent inhibitors.

Mutations within Ras proteins represent major drivers in human cancer. In this study, we report the structure-based design, synthesis, as well as biochemical and cellular evaluation of nucleotide-based covalent inhibitors for KRasG13C, an important oncogenic mutant of Ras that has not been successfully addressed in the past. Mass spectrometry experiments and kinetic studies reveal promising molecular properties of these covalent inhibitors, and X-ray crystallographic analysis has yielded the first reported crystal structures of KRasG13C covalently locked with these GDP analogues. Importantly, KRasG13C covalently modified with these inhibitors can no longer undergo SOS-catalysed nucleotide exchange. As a final proof-of-concept, we show that in contrast to KRasG13C, the covalently locked protein is unable to induce oncogenic signalling in cells, further highlighting the possibility of using nucleotide-based inhibitors with covalent warheads in KRasG13C-driven cancer.

Conformational transitions of the Spindly adaptor underlie its interaction with Dynein and Dynactin.

Cytoplasmic Dynein 1, or Dynein, is a microtubule minus end-directed motor. Dynein motility requires Dynactin and a family of activating adaptors that stabilize the Dynein-Dynactin complex and promote regulated interactions with cargo in space and time. How activating adaptors limit Dynein activation to specialized subcellular locales is unclear. Here, we reveal that Spindly, a mitotic Dynein adaptor at the kinetochore corona, exists natively in a closed conformation that occludes binding of Dynein-Dynactin to its CC1 box and Spindly motif. A structure-based analysis identified various mutations promoting an open conformation of Spindly that binds Dynein-Dynactin. A region of Spindly downstream from the Spindly motif and not required for cargo binding faces the CC1 box and stabilizes the intramolecular closed conformation. This region is also required for robust kinetochore localization of Spindly, suggesting that kinetochores promote Spindly activation to recruit Dynein. Thus, our work illustrates how specific Dynein activation at a defined cellular locale may require multiple factors.

Structure of the RZZ complex and molecular basis of Spindly-driven corona assembly at human kinetochores.

In metazoans, a ≈1 megadalton (MDa) multiprotein complex comprising the dynein-dynactin adaptor Spindly and the ROD-Zwilch-ZW10 (RZZ) complex is the building block of a fibrous biopolymer, the kinetochore fibrous corona. The corona assembles on mitotic kinetochores to promote microtubule capture and spindle assembly checkpoint (SAC) signaling. We report here a high-resolution cryo-EM structure that captures the essential features of the RZZ complex, including a farnesyl-binding site required for Spindly binding. Using a highly predictive in vitro assay, we demonstrate that the SAC kinase MPS1 is necessary and sufficient for corona assembly at supercritical concentrations of the RZZ-Spindly (RZZS) complex, and describe the molecular mechanism of phosphorylation-dependent filament nucleation. We identify several structural requirements for RZZS polymerization in rings and sheets. Finally, we identify determinants of kinetochore localization and corona assembly of Spindly. Our results describe a framework for the long-sought-for molecular basis of corona assembly on metazoan kinetochores.

In-Cell Double Electron-Electron Resonance at Nanomolar Protein Concentrations.

Electron paramagnetic resonance (EPR) spectroscopy is an established technique to site-specifically monitor conformational changes of spin-labeled biomolecules. Emerging in-cell EPR approaches aiming to address spin-labeled proteins in their native environment still struggle to reach a broad applicability and to target physiologically relevant protein concentrations. Here, we present a comparative in vitro and in-cell double electron-electron resonance (DEER) study demonstrating that nanomolar protein concentrations are at reach to measure distances up to 4.5 nm between protein sites carrying commercial gadolinium spin labels.

CDC20 assists its catalytic incorporation in the mitotic checkpoint complex.

Open (O) and closed (C) topologies of HORMA-domain proteins are respectively associated with inactive and active states of fundamental cellular pathways. The HORMA protein O-MAD2 converts to C-MAD2 upon binding CDC20. This is rate limiting for assembly of the mitotic checkpoint complex (MCC), the effector of a checkpoint required for mitotic fidelity. A catalyst assembled at kinetochores accelerates MAD2:CDC20 association through a poorly understood mechanism. Using a reconstituted SAC system, we discovered that CDC20 is an impervious substrate for which access to MAD2 requires simultaneous docking on several sites of the catalytic complex. Our analysis indicates that the checkpoint catalyst is substrate assisted and promotes MCC assembly through spatially and temporally coordinated conformational changes in both MAD2 and CDC20. This may define a paradigm for other HORMA-controlled systems.

Centriolar distal appendages activate the centrosome-PIDDosome-p53 signalling axis via ANKRD26.

Centrosome amplification results into genetic instability and predisposes cells to neoplastic transformation. Supernumerary centrosomes trigger p53 stabilization dependent on the PIDDosome (a multiprotein complex composed by PIDD1, RAIDD and Caspase-2), whose activation results in cleavage of p53's key inhibitor, MDM2. Here, we demonstrate that PIDD1 is recruited to mature centrosomes by the centriolar distal appendage protein ANKRD26. PIDDosome-dependent Caspase-2 activation requires not only PIDD1 centrosomal localization, but also its autoproteolysis. Following cytokinesis failure, supernumerary centrosomes form clusters, which appear to be necessary for PIDDosome activation. In addition, in the context of DNA damage, activation of the complex results from a p53-dependent elevation of PIDD1 levels independently of centrosome amplification. We propose that PIDDosome activation can in both cases be promoted by an ANKRD26-dependent local increase in PIDD1 concentration close to the centrosome. Collectively, these findings provide a paradigm for how centrosomes can contribute to cell fate determination by igniting a signalling cascade.

BUB1 and CENP-U, Primed by CDK1, Are the Main PLK1 Kinetochore Receptors in Mitosis.

Reflecting its pleiotropic functions, Polo-like kinase 1 (PLK1) localizes to various sub-cellular structures during mitosis. At kinetochores, PLK1 contributes to microtubule attachments and mitotic checkpoint signaling. Previous studies identified a wealth of potential PLK1 receptors at kinetochores, as well as requirements for various mitotic kinases, including BUB1, Aurora B, and PLK1 itself. Here, we combine ectopic localization, in vitro reconstitution, and kinetochore localization studies to demonstrate that most and likely all of the PLK1 is recruited through BUB1 in the outer kinetochore and centromeric protein U (CENP-U) in the inner kinetochore. BUB1 and CENP-U share a constellation of sequence motifs consisting of a putative PP2A-docking motif and two neighboring PLK1-docking sites, which, contingent on priming phosphorylation by cyclin-dependent kinase 1 and PLK1 itself, bind PLK1 and promote its dimerization. Our results rationalize previous observations and describe a unifying mechanism for recruitment of PLK1 to human kinetochores.

Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology.

Delivery of native or chemically modified recombinant proteins into mammalian cells shows promise for functional investigations and various technological applications, but concerns that sub-cellular localization and functional integrity of delivered proteins may be affected remain high. Here, we surveyed batch electroporation as a delivery tool for single polypeptides and multi-subunit protein assemblies of the kinetochore, a spatially confined and well-studied subcellular structure. After electroporation into human cells, recombinant fluorescent Ndc80 and Mis12 multi-subunit complexes exhibited native localization, physically interacted with endogenous binding partners, and functionally complemented depleted endogenous counterparts to promote mitotic checkpoint signaling and chromosome segregation. Farnesylation is required for kinetochore localization of the Dynein adaptor Spindly. In cells with chronically inhibited farnesyl transferase activity, in vitro farnesylation and electroporation of recombinant Spindly faithfully resulted in robust kinetochore localization. Our data show that electroporation is well-suited to deliver synthetic and chemically modified versions of functional proteins, and, therefore, constitutes a promising tool for applications in chemical and synthetic biology.

Cellular response upon proliferation in the presence of an active mitotic checkpoint.

Eukaryotic cells treated with microtubule-targeting agents activate the spindle assembly checkpoint to arrest in mitosis and prevent chromosome mis-segregation. A fraction of mitotically arrested cells overcomes the block and proliferates even under persistent checkpoint-activating conditions. Here, we asked what allows proliferation in such unfavourable conditions. We report that yeast cells are delayed in mitosis at each division, implying that their spindle assembly checkpoint remains responsive. The arrest causes their cell cycle to be elongated and results in a size increase. Growth saturates at mitosis and correlates with the repression of various factors involved in translation. Contrary to unperturbed cells, growth of cells with an active checkpoint requires Cdh1. This peculiar cell cycle correlates with global changes in protein expression whose signatures partly overlap with the environmental stress response. Hence, cells dividing with an active checkpoint develop recognisable specific traits that allow them to successfully complete cell division notwithstanding a constant mitotic checkpoint arrest. These properties distinguish them from unperturbed cells. Our observation may have implications for the identification of new therapeutic windows and targets in tumors.

Reconstitution of a 26-Subunit Human Kinetochore Reveals Cooperative Microtubule Binding by CENP-OPQUR and NDC80.

The approximately thirty core subunits of kinetochores assemble on centromeric chromatin containing the histone H3 variant CENP-A and connect chromosomes with spindle microtubules. The chromatin proximal 16-subunit CCAN (constitutive centromere associated network) creates a mechanically stable bridge between CENP-A and the kinetochore's microtubule-binding machinery, the 10-subunit KMN assembly. Here, we reconstituted a stoichiometric 11-subunit human CCAN core that forms when the CENP-OPQUR complex binds to a joint interface on the CENP-HIKM and CENP-LN complexes. The resulting CCAN particle is globular and connects KMN and CENP-A in a 26-subunit recombinant particle. The disordered, basic N-terminal tail of CENP-Q binds microtubules and promotes accurate chromosome alignment, cooperating with KMN in microtubule binding. The N-terminal basic tail of the NDC80 complex, the microtubule-binding subunit of KMN, can functionally replace the CENP-Q tail. Our work dissects the connectivity and architecture of CCAN and reveals unexpected functional similarities between CENP-OPQUR and the NDC80 complex.

The kinetochore proteins CENP-E and CENP-F directly and specifically interact with distinct BUB mitotic checkpoint Ser/Thr kinases.

The segregation of chromosomes during cell division relies on the function of the kinetochores, protein complexes that physically connect chromosomes with microtubules of the spindle. The metazoan proteins, centromere protein E (CENP-E) and CENP-F, are components of a fibrous layer of mitotic kinetochores named the corona. Several of their features suggest that CENP-E and CENP-F are paralogs: they are very large (comprising ∼2700 and 3200 residues, respectively), contain abundant predicted coiled-coil structures, are C-terminally prenylated, and are endowed with microtubule-binding sites at their termini. Moreover, CENP-E contains an ATP-hydrolyzing motor domain that promotes microtubule plus end-directed motion. Here, we show that both CENP-E and CENP-F are recruited to mitotic kinetochores independently of the main corona constituent, the Rod/Zwilch/ZW10 (RZZ) complex. We identified specific interactions of CENP-F and CENP-E with budding uninhibited by benzimidazole 1 (BUB1) and BUB1-related (BUBR1) mitotic checkpoint Ser/Thr kinases, respectively, paralogous proteins involved in mitotic checkpoint control and chromosome alignment. Whereas BUBR1 was dispensable for kinetochore localization of CENP-E, BUB1 was stringently required for CENP-F localization. Through biochemical reconstitution, we demonstrated that the CENP-E/BUBR1 and CENP-F/BUB1 interactions are direct and require similar determinants, a dimeric coiled-coil in CENP-E or CENP-F and a kinase domain in BUBR1 or BUB1. Our findings are consistent with the existence of structurally similar BUB1/CENP-F and BUBR1/CENP-E complexes, supporting the notion that CENP-E and CENP-F are evolutionarily related.

Mechanism and dynamics of INPP5E transport into and inside the ciliary compartment.

The inositol polyphosphate 5'-phosphatase E (INPP5E) localizes to cilia. We showed that the carrier protein phosphodiesterase 6 delta subunit (PDE6δ) mediates the sorting of farnesylated INPP5E into cilia due to high affinity binding and release by the ADP-ribosylation factor (Arf)-like protein Arl3·GTP. However, the dynamics of INPP5E transport into and inside the ciliary compartment are not fully understood. Here, we investigate the movement of INPP5E using live cell fluorescence microscopy and fluorescence recovery after photobleaching (FRAP) analysis. We show that PDE6δ and the dynein transport system are essential for ciliary sorting and entry of INPP5E. However, its innerciliary transport is regulated solely by the intraflagellar transport (IFT) system, independent from PDE6δ activity and INPP5E farnesylation. By contrast, movement of Arl3 into and within cilia occurs freely by diffusion and IFT-independently. The farnesylation defective INPP5E CaaX box mutant loses the exclusive ciliary localization. The accumulation of this mutant at centrioles after photobleaching suggests an affinity trap mechanism for ciliary entry, that in case of the wild type is overcome by the interaction with PDE6δ. Collectively, we postulate a three-step mechanism regulating ciliary localization of INPP5E, consisting of farnesylation- and PDE6δ-mediated targeting, INPP5E-PDE6δ complex diffusion into the cilium with transfer to the IFT system, and retention inside cilia.

Decoding the centromeric nucleosome through CENP-N.

Centromere protein (CENP) A, a histone H3 variant, is a key epigenetic determinant of chromosome domains known as centromeres. Centromeres nucleate kinetochores, multi-subunit complexes that capture spindle microtubules to promote chromosome segregation during mitosis. Two kinetochore proteins, CENP-C and CENP-N, recognize CENP-A in the context of a rare CENP-A nucleosome. Here, we reveal the structural basis for the exquisite selectivity of CENP-N for centromeres. CENP-N uses charge and space complementarity to decode the L1 loop that is unique to CENP-A. It also engages in extensive interactions with a 15-base pair segment of the distorted nucleosomal DNA double helix, in a position predicted to exclude chromatin remodelling enzymes. Besides CENP-A, stable centromere recruitment of CENP-N requires a coincident interaction with a newly identified binding motif on nucleosome-bound CENP-C. Collectively, our studies clarify how CENP-N and CENP-C decode and stabilize the non-canonical CENP-A nucleosome to enforce epigenetic centromere specification and kinetochore assembly.

BubR1 Promotes Bub3-Dependent APC/C Inhibition during Spindle Assembly Checkpoint Signaling.

The spindle assembly checkpoint (SAC) prevents premature sister chromatid separation during mitosis. Phosphorylation of unattached kinetochores by the Mps1 kinase promotes recruitment of SAC machinery that catalyzes assembly of the SAC effector mitotic checkpoint complex (MCC). The SAC protein Bub3 is a phospho-amino acid adaptor that forms structurally related stable complexes with functionally distinct paralogs named Bub1 and BubR1. A short motif ("loop") of Bub1, but not the equivalent loop of BubR1, enhances binding of Bub3 to kinetochore phospho-targets. Here, we asked whether the BubR1 loop directs Bub3 to different phospho-targets. The BubR1 loop is essential for SAC function and cannot be removed or replaced with the Bub1 loop. BubR1 loop mutants bind Bub3 and are normally incorporated in MCC in vitro but have reduced ability to inhibit the MCC target anaphase-promoting complex (APC/C), suggesting that BubR1:Bub3 recognition and inhibition of APC/C requires phosphorylation. Thus, small sequence differences in Bub1 and BubR1 direct Bub3 to different phosphorylated targets in the SAC signaling cascade.

Structure of the RZZ complex and molecular basis of its interaction with Spindly.

Kinetochores are macromolecular assemblies that connect chromosomes to spindle microtubules (MTs) during mitosis. The metazoan-specific ≈800-kD ROD-Zwilch-ZW10 (RZZ) complex builds a fibrous corona that assembles on mitotic kinetochores before MT attachment to promote chromosome alignment and robust spindle assembly checkpoint signaling. In this study, we combine biochemical reconstitutions, single-particle electron cryomicroscopy, cross-linking mass spectrometry, and structural modeling to build a complete model of human RZZ. We find that RZZ is structurally related to self-assembling cytosolic coat scaffolds that mediate membrane cargo trafficking, including Clathrin, Sec13-Sec31, and αß'ε-COP. We show that Spindly, a dynein adaptor, is related to BicD2 and binds RZZ directly in a farnesylation-dependent but membrane-independent manner. Through a targeted chemical biology approach, we identify ROD as the Spindly farnesyl receptor. Our results suggest that RZZ is dynein's cargo at human kinetochores.

Basis of catalytic assembly of the mitotic checkpoint complex.

In mitosis, for each daughter cell to inherit an accurate copy of the genome from the mother cell, sister chromatids in the mother cell must attach to microtubules emanating from opposite poles of the mitotic spindle, a process known as bi-orientation. A surveillance mechanism, termed the spindle assembly checkpoint (SAC), monitors the microtubule attachment process and can temporarily halt the separation of sister chromatids and the completion of mitosis until bi-orientation is complete. SAC failure results in abnormal chromosome numbers, termed aneuploidy, in the daughter cells, a hallmark of many tumours. The HORMA-domain-containing protein mitotic arrest deficient 2 (MAD2) is a subunit of the SAC effector mitotic checkpoint complex (MCC). Structural conversion from the open to the closed conformation of MAD2 is required for MAD2 to be incorporated into the MCC. In vitro, MAD2 conversion and MCC assembly take several hours, but in cells the SAC response is established in a few minutes. Here, to address this discrepancy, we reconstituted a near-complete SAC signalling system with purified components and monitored assembly of the MCC in real time. A marked acceleration in MAD2 conversion and MCC assembly was observed when monopolar spindle 1 (MPS1) kinase phosphorylated the MAD1-MAD2 complex, triggering it to act as the template for MAD2 conversion and therefore contributing to the establishment of a physical platform for MCC assembly. Thus, catalytic activation of the SAC depends on regulated protein-protein interactions that accelerate the spontaneous but rate-limiting conversion of MAD2 required for MCC assembly.

ARHGEF17 is an essential spindle assembly checkpoint factor that targets Mps1 to kinetochores.

To prevent genome instability, mitotic exit is delayed until all chromosomes are properly attached to the mitotic spindle by the spindle assembly checkpoint (SAC). In this study, we characterized the function of ARHGEF17, identified in a genome-wide RNA interference screen for human mitosis genes. Through a series of quantitative imaging, biochemical, and biophysical experiments, we showed that ARHGEF17 is essential for SAC activity, because it is the major targeting factor that controls localization of the checkpoint kinase Mps1 to the kinetochore. This mitotic function is mediated by direct interaction of the central domain of ARHGEF17 with Mps1, which is autoregulated by the activity of Mps1 kinase, for which ARHGEF17 is a substrate. This mitosis-specific role is independent of ARHGEF17's RhoGEF activity in interphase. Our study thus assigns a new mitotic function to ARHGEF17 and reveals the molecular mechanism for a key step in SAC establishment.

Requirement for PLK1 kinase activity in the maintenance of a robust spindle assembly checkpoint.

During mitotic arrest induced by microtubule targeting drugs, the weakening of the spindle assembly checkpoint (SAC) allows cells to progress through the cell cycle without chromosome segregation occurring. PLK1 kinase plays a major role in mitosis and emerging evidence indicates that PLK1 is also involved in establishing the checkpoint and maintaining SAC signalling. However, mechanistically, the role of PLK1 in the SAC is not fully understood, with several recent reports indicating that it can cooperate with either one of the major checkpoint kinases, Aurora B or MPS1. In this study, we assess the role of PLK1 in SAC maintenance. We find that in nocodazole-arrested U2OS cells, PLK1 activity is continuously required for maintaining Aurora B protein localisation and activity at kinetochores. Consistent with published data we find that upon PLK1 inhibition, phosphoThr3-H3, a marker of Haspin activity, is reduced. Intriguingly, Aurora B inhibition causes PLK1 to relocalise from kinetochores into fewer and much larger foci, possibly due to incomplete recruitment of outer kinetochore proteins. Importantly, PLK1 inhibition, together with partial inhibition of Aurora B, allows efficient SAC override to occur. This phenotype is more pronounced than the phenotype observed by combining the same PLK1 inhibitors with partial MPS1 inhibition. We also find that PLK1 inhibition does not obviously cooperate with Haspin inhibition to promote SAC override. These results indicate that PLK1 is directly involved in maintaining efficient SAC signalling, possibly by cooperating in a positive feedback loop with Aurora B, and that partially redundant mechanisms exist which reinforce the SAC.

A molecular basis for the differential roles of Bub1 and BubR1 in the spindle assembly checkpoint.

The spindle assembly checkpoint (SAC) monitors and promotes kinetochore-microtubule attachment during mitosis. Bub1 and BubR1, SAC components, originated from duplication of an ancestor gene. Subsequent sub-functionalization established subordination: Bub1, recruited first to kinetochores, promotes successive BubR1 recruitment. Because both Bub1 and BubR1 hetero-dimerize with Bub3, a targeting adaptor for phosphorylated kinetochores, the molecular basis for such sub-functionalization is unclear. We demonstrate that Bub1, but not BubR1, enhances binding of Bub3 to phosphorylated kinetochores. Grafting a short motif of Bub1 onto BubR1 promotes Bub1-independent kinetochore recruitment of BubR1. This gain-of-function BubR1 mutant cannot sustain a functional checkpoint. We demonstrate that kinetochore localization of BubR1 relies on direct hetero-dimerization with Bub1 at a pseudo-symmetric interface. This pseudo-symmetric interaction underpins a template-copy relationship crucial for kinetochore-microtubule attachment and SAC signaling. Our results illustrate how gene duplication and sub-functionalization shape the workings of an essential molecular network.

The pseudo GTPase CENP-M drives human kinetochore assembly.

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