The identification of crosslinks by mass spectrometry has recently been established as an integral part of the hybrid structural analysis of protein complexes and networks. The crosslinking analysis ...determines distance restraints between two covalently linked amino acids which are typically summarized in a table format that precludes the immediate and comprehensive interpretation of the topological data. xVis displays crosslinks in clear schematic representations in form of a circular, bar or network diagram. The interactive graphs indicate the linkage sites and identification scores, depict the spatial proximity of structurally and functionally annotated protein regions and the evolutionary conservation of amino acids and facilitate clustering of proteins into subcomplexes according to the crosslink density. Furthermore, xVis offers two options for the qualitative assessment of the crosslink identifications by filtering crosslinks according to identification scores or false discovery rates and by displaying the corresponding fragment ion spectrum of each crosslink for the manual validation of the mass spectrometric data. Our web server provides an easy-to-use tool for the fast topological and functional interpretation of distance information on protein complex architectures and for the evaluation of crosslink fragment ion spectra. xVis is available under a Creative Commons Attribution-ShareAlike 4.0 International license at http://xvis.genzentrum.lmu.de/.
Kinetochores are multisubunit complexes that assemble on centromeres to bind spindle microtubules and promote faithful chromosome segregation during cell division. A 16-subunit complex named the ...constitutive centromere-associated network (CCAN) creates the centromere-kinetochore interface. CENP-C, a CCAN subunit, is crucial for kinetochore assembly because it links centromeres with the microtubule-binding interface of kinetochores. The role of CENP-C in CCAN organization, on the other hand, had been incompletely understood. In this paper, we combined biochemical reconstitution and cellular investigations to unveil how CENP-C promotes kinetochore targeting of other CCAN subunits. The so-called PEST domain in the N-terminal half of CENP-C interacted directly with the four-subunit CCAN subcomplex CENP-HIKM. We identified crucial determinants of this interaction whose mutation prevented kinetochore localization of CENP-HIKM and of CENP-TW, another CCAN subcomplex. When considered together with previous observations, our data point to CENP-C as a blueprint for kinetochore assembly.
EB1 (end binding 1) proteins have emerged as central regulators of microtubule (MT) plus ends in all eukaryotes, but molecular mechanisms controlling the activity of these proteins are poorly ...understood. In this study, we show that the budding yeast EB1 protein Bim1p is regulated by Aurora B/Ipl1p-mediated multisite phosphorylation. Bim1p forms a stable complex with Ipl1p and is phosphorylated on a cluster of six Ser residues in the flexible linker connecting the calponin homology (CH) and EB1 domains. Using reconstitution of plus end tracking in vitro and total internal reflection fluorescence microscopy, we show that dimerization of Bim1p and the presence of the linker domain are both required for efficient tip tracking and that linker phosphorylation removes Bim1p from static and dynamic MTs. Bim1 phosphorylation occurs during anaphase in vivo, and it is required for normal spindle elongation kinetics and an efficient disassembly of the spindle midzone. Our results define a mechanism for the use and regulation of CH domains in an EB1 protein.
Transcription regulation in metazoans often involves promoter-proximal pausing of RNA polymerase (Pol) II, which requires the 4-subunit negative elongation factor (NELF). Here we discern the ...functional architecture of human NELF through X-ray crystallography, protein crosslinking, biochemical assays, and RNA crosslinking in cells. We identify a NELF core subcomplex formed by conserved regions in subunits NELF-A and NELF-C, and resolve its crystal structure. The NELF-AC subcomplex binds single-stranded nucleic acids in vitro, and NELF-C associates with RNA in vivo. A positively charged face of NELF-AC is involved in RNA binding, whereas the opposite face of the NELF-AC subcomplex binds NELF-B. NELF-B is predicted to form a HEAT repeat fold, also binds RNA in vivo, and anchors the subunit NELF-E, which is confirmed to bind RNA in vivo. These results reveal the three-dimensional architecture and three RNA-binding faces of NELF.
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.
Kinetochores are megadalton-sized protein complexes that mediate chromosome-microtubule interactions in eukaryotes. How kinetochore assembly is triggered specifically on centromeric chromatin is ...poorly understood. Here we use biochemical reconstitution experiments alongside genetic and structural analysis to delineate the contributions of centromere-associated proteins to kinetochore assembly in yeast. We show that the conserved kinetochore subunits Ame1(CENP-U) and Okp1(CENP-Q) form a DNA-binding complex that associates with the microtubule-binding KMN network via a short Mtw1 recruitment motif in the N terminus of Ame1. Point mutations in the Ame1 motif disrupt kinetochore function by preventing KMN assembly on chromatin. Ame1-Okp1 directly associates with the centromere protein C (CENP-C) homologue Mif2 to form a cooperative binding platform for outer kinetochore assembly. Our results indicate that the key assembly steps, CENP-A recognition and outer kinetochore recruitment, are executed through different yeast constitutive centromere-associated network subunits. This two-step mechanism may protect against inappropriate kinetochore assembly similar to rate-limiting nucleation steps used by cytoskeletal polymers.
Partitioning of the genome requires kinetochores, large protein complexes that mediate dynamic attachment of chromosomes to the spindle. Kinetochores contain two supramolecular protein assemblies. ...The ten-protein KMN network harbors key microtubule-binding sites in the Ndc80 complex and mediates assembly of checkpoint complexes via the KNL-1/Spc105 protein 1, 2. As KMN does not contact DNA directly, it relies on different centromere-binding proteins for recruitment and cell-cycle-dependent assembly. These proteins are collectively referred to as the CCAN (constitutive centromere-associated network) 2–4. The molecular mechanisms by which CCAN subunits associate, however, have remained incompletely defined. In particular, it is unclear how CCAN subunits facilitate the assembly of a microtubule-binding interface that contains multiple Ndc80 molecules bound to different receptors 5. Here, we dissect molecular mechanisms that underlie targeting of the CCAN subunit Cnn1/CENP-T to the sequence-determined point centromeres of budding yeast. Systematic quantitative mass spectrometry experiments reveal association dependencies within the yeast CCAN network. We show that evolutionarily conserved residues in the histone-fold domain of Cnn1 are required for the formation of a stable five-subunit CCAN subassembly with the Ctf3 complex. Cnn1 localizes in a Ctf3-dependent manner to the core of the yeast point centromere, overlapping with the yeast CENP-A protein Cse4. By arranging the N-terminal domains of the CCAN subunits Mcm16, Mcm22, and Cnn1 into close proximity, the Ctf3c-Cnn1-Wip1 complex configures a composite interaction site for two molecules of the Ndc80 complex. Our experiments show how cooperative assembly mechanisms organize the microtubule-binding interface of the kinetochore.
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•Subunit deletions reveal association dependencies in the budding yeast CCAN network•The Cnn1 histone-fold extension is required for assembly of a complex with Ctf3c•Cnn1 localizes to the core yeast centromere in a Ctf3-dependent manner•The Cnn1-Wip1-Ctf3c complex binds two Ndc80 molecules in a parallel configuration
Pekgöz Altunkaya, Malvezzi et al. investigate the assembly of the kinetochore, a large complex connecting chromosomes to the mitotic spindle. They identify protein-protein interactions involving histone-fold domains, which allow the binding of multiple molecules of the key microtubule-binding complex Ndc80 in order to generate robust attachments.
The bacterial type VI secretion system is a multicomponent molecular machine directed against eukaryotic host cells and competing bacteria. An intracellular contractile tubular structure that bears ...functional homology with bacteriophage tails is pivotal for ejection of pathogenic effectors. Here, we present the 6 Å cryoelectron microscopy structure of the contracted Vibrio cholerae tubule consisting of the proteins VipA and VipB. We localized VipA and VipB in the protomer and identified structural homology between the C-terminal segment of VipB and the tail-sheath protein of T4 phages. We propose that homologous segments in VipB and T4 phages mediate tubule contraction. We show that in type VI secretion, contraction leads to exposure of the ClpV recognition motif, which is embedded in the type VI-specific four-helix-bundle N-domain of VipB. Disaggregation of the tubules by the AAA+ protein ClpV and recycling of the VipA/B subunits are thereby limited to the contracted state.
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•Helical arrangement of VipA/B tubules resembles that of contracted phage tails•Homologous VipB and viral tail sheath core regions stabilize the contracted tubule•VipA and a unique VipB N-terminal domain adapt the T6SS for bacterial secretion•Exposed recognition motif mediates contraction-state-specific disassembly by ClpV
Type VI secretion systems (T6SSs) are involved in bacterial competition and virulence. Here, Kube et al. report the structure of the Vibrio cholerae VipA/B T6SS tubule in its contracted state. The overall architecture of the tubule resembles bacteriophage tails. However, further similarities are restricted to the inner-architecture-defining domains of VipB. The structure indicates a mechanism for contraction-state-specific recycling by a unique N-terminal domain, which is only accessible in the contracted state.
Kinetochores are macromolecular protein assemblies at centromeres that mediate accurate chromosome segregation during cell division. The outer kinetochore KNL1SPC105, MIS12MTW1, and NDC80NDC80 ...complexes assemble the KMN network, which harbors the sites of microtubule binding and spindle assembly checkpoint signaling. The buildup of the KMN network that transmits microtubule pulling forces to budding yeast point centromeres is poorly understood. Here, we identify 225 inter-protein crosslinks by mass spectrometry on KMN complexes isolated from Saccharomyces cerevisiae that delineate the KMN subunit connectivity for outer kinetochore assembly. C-Terminal motifs of Nsl1 and Mtw1 recruit the SPC105 complex through Kre28, and both motifs aid tethering of the NDC80 complex by the previously reported Dsn1 C terminus. We show that a hub of three C-terminal MTW1 subunit motifs mediates the cooperative stabilization of the KMN network, which is augmented by a direct NDC80-SPC105 association.
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•XL-MS of native kinetochore complexes identifies KMN subunit connectivity in yeast•Two C-terminal MTW1 subunit motifs recruit SPC105 complex through Kre28•C-terminal MTW1 motifs cooperatively stabilize the KMN network•Cooperative assembly of the KMN network is enhanced by NDC80-SPC105 association
The kinetochore links chromosomes to spindle microtubules and has to withstand the pulling forces of depolymerizing microtubules to segregate chromosomes during cell division. Using chemical crosslinking and mass spectrometry, Ghodgaonkar-Steger et al. identify the protein motifs that cooperatively stabilize the 10-subunit outer kinetochore network that forms the microtubule binding site.
Oscillating cyclin-dependent kinase 1 (Cdk1) activity is the major regulator of cell-cycle progression 1, whereas the Aurora B kinase, as part of the chromosome passenger complex (CPC), controls ...critical aspects of mitosis such as chromosome condensation and biorientation on the spindle 2. How these kinases mechanistically coordinate their important functions is only partially understood. Here, using budding yeast, we identify a regulatory mechanism by which the Cdk1 kinase Cdc28 directly controls the Aurora kinase Ipl1. We show that Cdk1 phosphorylates Ipl1 on two serine residues in the N-terminal domain, thereby suppressing its association with the microtubule plus-end tracking protein Bim1 until the onset of anaphase. Failure to phosphorylate Ipl1 leads to its premature targeting to the metaphase spindle and results in constitutive Bim1 phosphorylation, which is normally restricted to anaphase. Cells expressing an Ipl1-Sli15 complex that cannot be phosphorylated by Cdk1 display a severe growth defect. Our work shows that Ipl1/Aurora is not only the catalytic subunit of the CPC but also an important regulatory target that allows Cdk1 to coordinate chromosome biorientation with spindle morphogenesis.
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► Ipl1/Aurora kinase is an SxIP motif containing plus-end tracking protein (+TIP) ► Cdk1 phosphorylation prevents Ipl1-Bim1 association until anaphase onset ► Cdk1 phosphorylation allows cell-cycle-specific phosphorylation of Bim1 ► Cdk1 phosphorylation of the Ipl1-Sli15 complex is critical for viability
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