RESUMEN
Microtubule (MT) filaments, composed of α/ß-tubulin dimers, are fundamental to cellular architecture, function and organismal development. They are nucleated from MT organizing centers by the evolutionarily conserved γ-tubulin ring complex (γTuRC). However, the molecular mechanism of nucleation remains elusive. Here we used cryo-electron tomography to determine the structure of the native γTuRC capping the minus end of a MT in the context of enriched budding yeast spindles. In our structure, γTuRC presents a ring of γ-tubulin subunits to seed nucleation of exclusively 13-protofilament MTs, adopting an active closed conformation to function as a perfect geometric template for MT nucleation. Our cryo-electron tomography reconstruction revealed that a coiled-coil protein staples the first row of α/ß-tubulin of the MT to alternating positions along the γ-tubulin ring of γTuRC. This positioning of α/ß-tubulin onto γTuRC suggests a role for the coiled-coil protein in augmenting γTuRC-mediated MT nucleation. Based on our results, we describe a molecular model for budding yeast γTuRC activation and MT nucleation.
Asunto(s)
Microscopía por Crioelectrón , Microtúbulos , Modelos Moleculares , Saccharomyces cerevisiae , Huso Acromático , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/ultraestructura , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Microtúbulos/química , Huso Acromático/metabolismo , Huso Acromático/ultraestructura , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestructura , Tomografía con Microscopio Electrónico , Conformación Proteica , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/ultraestructuraRESUMEN
Condensin is a structural maintenance of chromosomes (SMC) complex family member thought to build mitotic chromosomes by DNA loop extrusion. However, condensin variants unable to extrude loops, yet proficient in chromosome formation, were recently described. Here, we explore how condensin might alternatively build chromosomes. Using bulk biochemical and single-molecule experiments with purified fission yeast condensin, we observe that individual condensins sequentially and topologically entrap two double-stranded DNAs (dsDNAs). Condensin loading transitions through a state requiring DNA bending, as proposed for the related cohesin complex. While cohesin then favors the capture of a second single-stranded DNA (ssDNA), second dsDNA capture emerges as a defining feature of condensin. We provide complementary in vivo evidence for DNA-DNA capture in the form of condensin-dependent chromatin contacts within, as well as between, chromosomes. Our results support a "diffusion capture" model in which condensin acts in mitotic chromosome formation by sequential dsDNA-dsDNA capture.
Asunto(s)
Proteínas de Unión al ADN , Schizosaccharomyces , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/química , ADN/genética , Cromosomas , Proteínas de Ciclo Celular/genética , Schizosaccharomyces/genética , MitosisRESUMEN
The chromosomal cohesin complex establishes sister chromatid cohesion during S phase, which forms the basis for faithful segregation of DNA replication products during cell divisions. Cohesion establishment is defective in the absence of either of three non-essential Saccharomyces cerevisiae replication fork components Tof1-Csm3 and Mrc1. Here, we investigate how these conserved factors contribute to cohesion establishment. Tof1-Csm3 and Mrc1 serve known roles during DNA replication, including replication checkpoint signaling, securing replication fork speed, as well as recruiting topoisomerase I and the histone chaperone FACT. By modulating each of these functions independently, we rule out that one of these known replication roles explains the contribution of Tof1-Csm3 and Mrc1 to cohesion establishment. Instead, using purified components, we reveal direct and multipronged protein interactions of Tof1-Csm3 and Mrc1 with the cohesin complex. Our findings open the possibility that a series of physical interactions between replication fork components and cohesin facilitate successful establishment of sister chromatid cohesion during DNA replication.
Asunto(s)
Replicación del ADN , Proteínas de Saccharomyces cerevisiae , Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cromátides/metabolismo , CohesinasRESUMEN
DNA interstrand cross-links are tumor-inducing lesions that block DNA replication and transcription. When cross-links are detected at stalled replication forks, ATR kinase phosphorylates FANCI, which stimulates monoubiquitination of the FANCD2-FANCI clamp by the Fanconi anemia core complex. Monoubiquitinated FANCD2-FANCI is locked onto DNA and recruits nucleases that mediate DNA repair. However, it remains unclear how phosphorylation activates this pathway. Here, we report structures of FANCD2-FANCI complexes containing phosphomimetic FANCI. We observe that, unlike wild-type FANCD2-FANCI, the phosphomimetic complex closes around DNA, independent of the Fanconi anemia core complex. The phosphomimetic mutations do not substantially alter DNA binding but instead destabilize the open state of FANCD2-FANCI and alter its conformational dynamics. Overall, our results demonstrate that phosphorylation primes the FANCD2-FANCI clamp for ubiquitination, showing how multiple posttranslational modifications are coordinated to control DNA repair.
Asunto(s)
Anemia de Fanconi , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , ADN/metabolismo , Daño del ADN , Reparación del ADN , Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Humanos , Polinucleótido 5'-Hidroxil-Quinasa/genética , Polinucleótido 5'-Hidroxil-Quinasa/metabolismo , UbiquitinaciónRESUMEN
The lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal and spatial control. PI3P is generated by two complexes that both contain the lipid kinase VPS34: complex II on endosomes (VPS34/VPS15/Beclin 1/UVRAG), and complex I on autophagosomes (VPS34/VPS15/Beclin 1/ATG14L). The endosomal GTPase Rab5 binds complex II, but the mechanism of VPS34 activation by Rab5 has remained elusive, and no GTPase is known to bind complex I. Here we show that Rab5a-GTP recruits endocytic complex II to membranes and activates it by binding between the VPS34 C2 and VPS15 WD40 domains. Electron cryotomography of complex II on Rab5a-decorated vesicles shows that the VPS34 kinase domain is released from inhibition by VPS15 and hovers over the lipid bilayer, poised for catalysis. We also show that the GTPase Rab1a, which is known to be involved in autophagy, recruits and activates the autophagy-specific complex I, but not complex II. Both Rabs bind to the same VPS34 interface but in a manner unique for each. These findings reveal how VPS34 complexes are activated on membranes by specific Rab GTPases and how they are recruited to unique cellular locations.
Asunto(s)
Membrana Celular/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/química , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Proteínas de Unión al GTP rab1/química , Proteínas de Unión al GTP rab1/metabolismo , Proteínas de Unión al GTP rab5/química , Proteínas de Unión al GTP rab5/metabolismo , Beclina-1/química , Beclina-1/genética , Beclina-1/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/genética , Endosomas/metabolismo , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Estructura Secundaria de Proteína , Tomografía , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína de Clasificación Vacuolar VPS15/química , Proteína de Clasificación Vacuolar VPS15/genética , Proteína de Clasificación Vacuolar VPS15/metabolismo , Proteínas de Unión al GTP rab1/genética , Proteínas de Unión al GTP rab5/genéticaRESUMEN
The main force generators in eukaryotic cilia and flagella are axonemal outer dynein arms (ODAs). During ciliogenesis, these ~1.8-megadalton complexes are assembled in the cytoplasm and targeted to cilia by an unknown mechanism. Here, we used the ciliate Tetrahymena to identify two factors (Q22YU3 and Q22MS1) that bind ODAs in the cytoplasm and are required for ODA delivery to cilia. Q22YU3, which we named Shulin, locked the ODA motor domains into a closed conformation and inhibited motor activity. Cryo-electron microscopy revealed how Shulin stabilized this compact form of ODAs by binding to the dynein tails. Our findings provide a molecular explanation for how newly assembled dyneins are packaged for delivery to the cilia.
Asunto(s)
Dineínas Axonemales/metabolismo , Cilios/metabolismo , Proteínas Protozoarias/metabolismo , Tetrahymena thermophila/fisiología , Dineínas Axonemales/química , Dineínas Axonemales/genética , Microscopía por Crioelectrón , Citoplasma/metabolismo , Técnicas de Silenciamiento del Gen , Procesamiento de Imagen Asistido por Computador , Microtúbulos/fisiología , Modelos Moleculares , Movimiento , Unión Proteica , Conformación Proteica , Dominios Proteicos , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Tetrahymena thermophila/genéticaRESUMEN
Cellular RNA polymerases (RNAPs) can become trapped on DNA or RNA, threatening genome stability and limiting free enzyme pools, but how RNAP recycling into active states is achieved remains elusive. In Bacillus subtilis, the RNAP δ subunit and NTPase HelD have been implicated in RNAP recycling. We structurally analyzed Bacillus subtilis RNAP-δ-HelD complexes. HelD has two long arms: a Gre cleavage factor-like coiled-coil inserts deep into the RNAP secondary channel, dismantling the active site and displacing RNA, while a unique helical protrusion inserts into the main channel, prying the ß and ß' subunits apart and, aided by δ, dislodging DNA. RNAP is recycled when, after releasing trapped nucleic acids, HelD dissociates from the enzyme in an ATP-dependent manner. HelD abundance during slow growth and a dimeric (RNAP-δ-HelD)2 structure that resembles hibernating eukaryotic RNAP I suggest that HelD might also modulate active enzyme pools in response to cellular cues.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Nucleósido-Trifosfatasa/metabolismo , Subunidades de Proteína/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/química , Dominio Catalítico , ARN Polimerasas Dirigidas por ADN/química , Modelos Moleculares , Nucleósido-Trifosfatasa/química , Multimerización de Proteína , Subunidades de Proteína/químicaRESUMEN
Despite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here we combine biochemical approaches and cryoelectron microscopy (cryo-EM) to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring. Building on this structural framework, we design experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an N-terminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against the ATPase gate. ATP hydrolysis will lead to ATPase gate opening to complete DNA entry. Whether DNA loading is successful or results in loop extrusion might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA.
Asunto(s)
Proteínas de Ciclo Celular/ultraestructura , Cromátides/ultraestructura , Proteínas Cromosómicas no Histona/ultraestructura , ADN/ultraestructura , Intercambio de Cromátides Hermanas/genética , Adenosina Trifosfatasas/genética , Proteínas de Ciclo Celular/genética , Cromátides/genética , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica/genética , Microscopía por Crioelectrón , ADN/genética , Conformación de Ácido Nucleico , Conformación Proteica , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestructura , CohesinasRESUMEN
Vertebrate DNA crosslink repair excises toxic replication-blocking DNA crosslinks. Numerous factors involved in crosslink repair have been identified, and mutations in their corresponding genes cause Fanconi anemia (FA). A key step in crosslink repair is monoubiquitination of the FANCD2-FANCI heterodimer, which then recruits nucleases to remove the DNA lesion. Here, we use cryo-EM to determine the structures of recombinant chicken FANCD2 and FANCI complexes. FANCD2-FANCI adopts a closed conformation when the FANCD2 subunit is monoubiquitinated, creating a channel that encloses double-stranded DNA (dsDNA). Ubiquitin is positioned at the interface of FANCD2 and FANCI, where it acts as a covalent molecular pin to trap the complex on DNA. In contrast, isolated FANCD2 is a homodimer that is unable to bind DNA, suggestive of an autoinhibitory mechanism that prevents premature activation. Together, our work suggests that FANCD2-FANCI is a clamp that is locked onto DNA by ubiquitin, with distinct interfaces that may recruit other DNA repair factors.
Asunto(s)
Reparación del ADN , ADN/química , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/química , Ubiquitina/química , Animales , Sitios de Unión , Pollos , Microscopía por Crioelectrón , Cristalografía por Rayos X , ADN/genética , ADN/metabolismo , Daño del ADN , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Expresión Génica , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Ubiquitina/genética , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
We present a concise workflow to enhance the mass spectrometric detection of crosslinked peptides by introducing sequential digestion and the crosslink identification software xiSEARCH. Sequential digestion enhances peptide detection by selective shortening of long tryptic peptides. We demonstrate our simple 12-fraction protocol for crosslinked multi-protein complexes and cell lysates, quantitative analysis, and high-density crosslinking, without requiring specific crosslinker features. This overall approach reveals dynamic protein-protein interaction sites, which are accessible, have fundamental functional relevance and are therefore ideally suited for the development of small molecule inhibitors.
Asunto(s)
Espectrometría de Masas/métodos , Mapeo de Interacción de Proteínas/métodos , Proteínas/química , Proteómica/métodos , Citosol/química , Humanos , Células K562 , Modelos Moleculares , Fragmentos de Péptidos/química , Conformación Proteica , Programas InformáticosRESUMEN
Quantitative cross-linking/mass spectrometry (QCLMS/QXL-MS) probes structural changes of proteins in solution. This method has revealed induced conformational changes, composition shifts in conformational ensembles and changes in protein interactions. It illuminates different structural states of proteins or protein complexes by comparing which residue pairs can be cross-linked in these states. Cross-links provide information about structural changes that may be inaccessible by alternative technologies. Small local conformational changes affect relative abundances of nearby cross-links, whereas larger conformational changes cause new cross-links to be formed. Distinguishing between minor and major changes requires a robust analysis based on carefully selected replicates and, when using isotope-labeled cross-linkers, replicated analysis with a permutated isotope-labeling scheme. A label-free workflow allows for application of a wide range of cross-linking chemistries and enables parallel comparison of multiple conformations. In this protocol, we demonstrate both label-free and isotope-labeled cross-linker-based workflows using the cross-linker bis(sulfosuccinimidyl)suberate (BS3). The software XiSearch, developed by our group, is used to identify cross-linked residue pairs, although the workflow is not limited to this search software. The open-access software Skyline is used for automated quantitation. Note that additional manual correction greatly enhances quantitation accuracy. The protocol has been applied to purified multi-protein complexes but is not necessarily limited to that level of sample complexity. Optimizing the cross-linker/protein ratio and fractionating peptides increase the data density of quantified cross-links, and thus the resolution of QCLMS. The entire procedure takes ~1-3 weeks.
Asunto(s)
Reactivos de Enlaces Cruzados/química , Espectrometría de Masas/métodos , Péptidos/análisis , Proteínas/química , Succinimidas/química , Secuencia de Aminoácidos , Humanos , Marcaje Isotópico/métodos , Espectrometría de Masas/instrumentación , Isótopos de Oxígeno/química , Péptidos/química , Conformación Proteica , Proteínas/aislamiento & purificación , Proteolisis , Programas Informáticos , Tripsina/química , Flujo de TrabajoRESUMEN
The dynamics of protein structures and their interactions are responsible for many cellular processes. The rearrangements and interactions of proteins, which are often transient, occur in solution and may require a biological environment that is difficult to maintain in traditional structural biological approaches. Quantitative crosslinking/mass spectrometry (QCLMS) has emerged as an excellent method to fill this gap. Numerous recent applications of the technique have demonstrated that protein dynamics can now be studied in solution at sufficient resolution to gain valuable biological insights, suggesting that extending these investigations to native environments is possible. These breakthroughs have been based on the maturation of CLMS at large, and its recent fusion with quantitative proteomics. We provide here an overview of the current state of the technique, the available workflows and their applications, and remaining challenges.
Asunto(s)
Reactivos de Enlaces Cruzados/metabolismo , Espectrometría de Masas/métodos , Conformación Proteica , Proteínas/química , Proteínas/metabolismo , Animales , Humanos , ProteómicaRESUMEN
The hetero-octameric protein complex, Augmin, recruits γ-Tubulin ring complex (γ-TuRC) to pre-existing microtubules (MTs) to generate branched MTs during mitosis, facilitating robust spindle assembly. However, despite a recent partial reconstitution of the human Augmin complex in vitro, the molecular basis of this recruitment remains unclear. Here, we used immuno-affinity purification of in vivo Augmin from Drosophila and cross-linking/mass spectrometry to identify distance restraints between residues within the eight Augmin subunits in the absence of any other structural information. The results allowed us to predict potential interfaces between Augmin and γ-TuRC. We tested these predictions biochemically and in the Drosophila embryo, demonstrating that specific regions of the Augmin subunits, Dgt3, Dgt5 and Dgt6 all directly bind the γ-TuRC protein, Dgp71WD, and are required for the accumulation of γ-TuRC, but not Augmin, to the mitotic spindle. This study therefore substantially increases our understanding of the molecular mechanisms underpinning MT-dependent MT nucleation.
RESUMEN
We present a baculovirus-based protein engineering method that enables site-specific introduction of unique functionalities in a eukaryotic protein complex recombinantly produced in insect cells. We demonstrate the versatility of this efficient and robust protein production platform, 'MultiBacTAG', (i) for the fluorescent labeling of target proteins and biologics using click chemistries, (ii) for glycoengineering of antibodies, and (iii) for structure-function studies of novel eukaryotic complexes using single-molecule Förster resonance energy transfer as well as site-specific crosslinking strategies.
Asunto(s)
Proteínas Fluorescentes Verdes/biosíntesis , Complejos Multiproteicos/biosíntesis , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/biosíntesis , Proteínas Virales/biosíntesis , Animales , Baculoviridae/genética , Baculoviridae/metabolismo , Técnicas de Cultivo de Célula , Transferencia Resonante de Energía de Fluorescencia/métodos , Código Genético , Vectores Genéticos , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Humanos , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Plásmidos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Células Sf9 , Spodoptera , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
The slow but spontaneous and ubiquitous formation of C3(H2O), the hydrolytic and conformationally rearranged product of C3, initiates antibody-independent activation of the complement system that is a key first line of antimicrobial defense. The structure of C3(H2O) has not been determined. Here we subjected C3(H2O) to quantitative cross-linking/mass spectrometry (QCLMS). This revealed details of the structural differences and similarities between C3(H2O) and C3, as well as between C3(H2O) and its pivotal proteolytic cleavage product, C3b, which shares functionally similarity with C3(H2O). Considered in combination with the crystal structures of C3 and C3b, the QCMLS data suggest that C3(H2O) generation is accompanied by the migration of the thioester-containing domain of C3 from one end of the molecule to the other. This creates a stable C3b-like platform able to bind the zymogen, factor B, or the regulator, factor H. Integration of available crystallographic and QCLMS data allowed the determination of a 3D model of the C3(H2O) domain architecture. The unique arrangement of domains thus observed in C3(H2O), which retains the anaphylatoxin domain (that is excised when C3 is enzymatically activated to C3b), can be used to rationalize observed differences between C3(H2O) and C3b in terms of complement activation and regulation.
Asunto(s)
Complemento C3/química , Complemento C3b/química , Espectrometría de Masas/métodos , Reactivos de Enlaces Cruzados , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Conformación Proteica , Dominios ProteicosRESUMEN
The conceptually simple step from cross-linking/mass spectrometry (CLMS) to quantitative cross-linking/mass spectrometry (QCLMS) is compounded by technical challenges. Currently, quantitative proteomics software is tightly integrated with the protein identification workflow. This prevents automatically quantifying other m/z features in a targeted manner including those associated with cross-linked peptides. Here we present a new release of MaxQuant that permits starting the quantification process from an m/z feature list. Comparing the automated quantification to a carefully manually curated test set of cross-linked peptides obtained by cross-linking C3 and C3b with BS(3) and isotope-labeled BS(3)-d4 revealed a number of observations: (1) Fully automated process using MaxQuant can quantify cross-links in our reference data set with 68% recall rate and 88% accuracy. (2) Hidden quantification errors can be converted into exposed failures by label-swap replica, which makes label-swap replica an essential part of QCLMS. (3) Cross-links that failed during automated quantification can be recovered by semi-automated re-quantification. The integrated workflow of MaxQuant and semi-automated assessment provides the maximum of quantified cross-links. In contrast, work on larger data sets or by less experienced users will benefit from full automation in MaxQuant.
Asunto(s)
Reactivos de Enlaces Cruzados/química , Proteómica/métodos , Marcaje Isotópico , Espectrometría de Masas/métodos , Programas InformáticosRESUMEN
Most short-lived eukaryotic proteins are degraded by the proteasome. A proteolytic core particle (CP) capped by regulatory particles (RPs) constitutes the 26S proteasome complex. RP biogenesis culminates with the joining of two large subcomplexes, the lid and base. In yeast and mammals, the lid appears to assemble completely before attaching to the base, but how this hierarchical assembly is enforced has remained unclear. Using biochemical reconstitutions, quantitative cross-linking/mass spectrometry, and electron microscopy, we resolve the mechanistic basis for the linkage between lid biogenesis and lid-base joining. Assimilation of the final lid subunit, Rpn12, triggers a large-scale conformational remodeling of the nascent lid that drives RP assembly, in part by relieving steric clash with the base. Surprisingly, this remodeling is triggered by a single Rpn12 α helix. Such assembly-coupled conformational switching is reminiscent of viral particle maturation and may represent a commonly used mechanism to enforce hierarchical assembly in multisubunit complexes.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Escherichia coli/metabolismo , Espectrometría de Masas , Microscopía Electrónica , Modelos Moleculares , Estructura Secundaria de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
In an attempt to evade annihilation by the vertebrate complement system, many microbes capture factor H (FH), the key soluble complement-regulating protein in human plasma. However, FH is normally an active complement suppressor exclusively on self-surfaces and this selective action of FH is pivotal to self versus non-self discrimination by the complement system. We investigated whether the bacterially captured FH becomes functionally enhanced and, if so, how this is achieved at a structural level. We found, using site-directed and truncation mutagenesis, surface plasmon resonance, nuclear magnetic resonance spectroscopy, and cross-linking and mass spectrometry, that the N-terminal domain of Streptococcus pneumoniae protein PspC (PspCN) not only binds FH extraordinarily tightly but also holds it in a previously uncharacterized conformation. Functional enhancement arises from exposure of a C-terminal cryptic second binding site in FH for C3b, the activation-specific fragment of the pivotal complement component, C3. This conformational change of FH doubles its affinity for C3b and increases 5-fold its ability to accelerate decay of the binary enzyme (C3bBb) responsible for converting C3 to C3b in an amplification loop. Despite not sharing critical FH-binding residues, PspCNs from D39 and Tigr4 S. pneumoniae exhibit similar FH-anchoring and enhancing properties. We propose that these bacterial proteins mimic molecular markers of self-surfaces, providing a compelling hypothesis for how FH prevents complement-mediated injury to host tissue while lacking efficacy on virtually all other surfaces. In hemolysis assays with 2-aminoethylisothiouronium bromide-treated erythrocytes that recapitulate paroxysmal nocturnal hemoglobinuria, PspCN enhanced protection of cells by FH, suggesting a new paradigm for therapeutic complement suppression.
Asunto(s)
Proteínas Bacterianas/química , Complemento C3b/química , Factor H de Complemento/química , Streptococcus pneumoniae/química , Proteínas Bacterianas/inmunología , Complemento C3b/inmunología , Factor H de Complemento/inmunología , Hemoglobinuria Paroxística/inmunología , Humanos , Estructura Terciaria de Proteína , Streptococcus pneumoniae/inmunologíaRESUMEN
Paralogs NDE1 (nuclear distribution element 1) and NDEL1 (NDE-like 1) are essential for mitosis and neurodevelopment. Both proteins are predicted to have similar structures, based upon high sequence similarity, and they co-complex in mammalian cells. X-ray diffraction studies and homology modeling suggest that their N-terminal regions (residues 8-167) adopt continuous, extended α-helical coiled-coil structures, but no experimentally derived information on the structure of their C-terminal regions or the architecture of the full-length proteins is available. In the case of NDE1, no biophysical data exists. Here we characterize the structural architecture of both full-length proteins utilizing negative stain electron microscopy along with our established paradigm of chemical cross-linking followed by tryptic digestion, mass spectrometry, and database searching, which we enhance using isotope labeling for mixed NDE1-NDEL1. We determined that full-length NDE1 forms needle-like dimers and tetramers in solution, similar to crystal structures of NDEL1, as well as chain-like end-to-end polymers. The C-terminal domain of each protein, required for interaction with key protein partners dynein and DISC1 (disrupted-in-schizophrenia 1), includes a predicted disordered region that allows a bent back structure. This facilitates interaction of the C-terminal region with the N-terminal coiled-coil domain and is in agreement with previous results showing N- and C-terminal regions of NDEL1 and NDE1 cooperating in dynein interaction. It sheds light on recently identified mutations in the NDE1 gene that cause truncation of the encoded protein. Additionally, analysis of mixed NDE1-NDEL1 complexes demonstrates that NDE1 and NDEL1 can interact directly.
Asunto(s)
Proteínas Portadoras/química , Proteínas Asociadas a Microtúbulos/química , Modelos Moleculares , Pliegue de Proteína , Multimerización de Proteína/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Estructura Cuaternaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Despite many decades of study, mitotic chromosome structure and composition remain poorly characterized. Here, we have integrated quantitative proteomics with bioinformatic analysis to generate a series of independent classifiers that describe the approximately 4,000 proteins identified in isolated mitotic chromosomes. Integrating these classifiers by machine learning uncovers functional relationships between protein complexes in the context of intact chromosomes and reveals which of the approximately 560 uncharacterized proteins identified here merits further study. Indeed, of 34 GFP-tagged predicted chromosomal proteins, 30 were chromosomal, including 13 with centromere-association. Of 16 GFP-tagged predicted nonchromosomal proteins, 14 were confirmed to be nonchromosomal. An unbiased analysis of the whole chromosome proteome from genetic knockouts of kinetochore protein Ska3/Rama1 revealed that the APC/C and RanBP2/RanGAP1 complexes depend on the Ska complex for stable association with chromosomes. Our integrated analysis predicts that up to 97 new centromere-associated proteins remain to be discovered in our data set.