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2.
EMBO Rep ; 22(7): e52242, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34013668

RESUMO

During metaphase, in response to improper kinetochore-microtubule attachments, the spindle assembly checkpoint (SAC) activates the mitotic checkpoint complex (MCC), an inhibitor of the anaphase-promoting complex/cyclosome (APC/C). This process is orchestrated by the kinase Mps1, which initiates the assembly of the MCC onto kinetochores through a sequential phosphorylation-dependent signalling cascade. The Mad1-Mad2 complex, which is required to catalyse MCC formation, is targeted to kinetochores through a direct interaction with the phosphorylated conserved domain 1 (CD1) of Bub1. Here, we present the crystal structure of the C-terminal domain of Mad1 (Mad1CTD ) bound to two phosphorylated Bub1CD1 peptides at 1.75 Å resolution. This interaction is mediated by phosphorylated Bub1 Thr461, which not only directly interacts with Arg617 of the Mad1 RLK (Arg-Leu-Lys) motif, but also directly acts as an N-terminal cap to the CD1 α-helix dipole. Surprisingly, only one Bub1CD1 peptide binds to the Mad1 homodimer in solution. We suggest that this stoichiometry is due to inherent asymmetry in the coiled-coil of Mad1CTD and has implications for how the Mad1-Bub1 complex at kinetochores promotes efficient MCC assembly.


Assuntos
Proteínas de Ciclo Celular , Cinetocoros , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Cinetocoros/metabolismo , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Fosforilação , Transdução de Sinais , Fuso Acromático/metabolismo
3.
J Virol ; 95(15): e0020321, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-33963055

RESUMO

The majority of SARS-CoV-2 vaccines in use or advanced development are based on the viral spike protein (S) as their immunogen. S is present on virions as prefusion trimers in which the receptor binding domain (RBD) is stochastically open or closed. Neutralizing antibodies have been described against both open and closed conformations. The long-term success of vaccination strategies depends upon inducing antibodies that provide long-lasting broad immunity against evolving SARS-CoV-2 strains. Here, we have assessed the results of immunization in a mouse model using an S protein trimer stabilized in the closed state to prevent full exposure of the receptor binding site and therefore interaction with the receptor. We compared this with other modified S protein constructs, including representatives used in current vaccines. We found that all trimeric S proteins induced a T cell response and long-lived, strongly neutralizing antibody responses against 2019 SARS-CoV-2 and variants of concern P.1 and B.1.351. Notably, the protein binding properties of sera induced by the closed spike differed from those induced by standard S protein constructs. Closed S proteins induced more potent neutralizing responses than expected based on the degree to which they inhibit interactions between the RBD and ACE2. These observations suggest that closed spikes recruit different, but equally potent, immune responses than open spikes and that this is likely to include neutralizing antibodies against conformational epitopes present in the closed conformation. We suggest that closed spikes, together with their improved stability and storage properties, may be a valuable component of refined, next-generation vaccines. IMPORTANCE Vaccines in use against SARS-CoV-2 induce immune responses against the spike protein. There is intense interest in whether the antibody response induced by vaccines will be robust against new variants, as well as in next-generation vaccines for use in previously infected or immunized individuals. We assessed the use as an immunogen of a spike protein engineered to be conformationally stabilized in the closed state where the receptor binding site is occluded. Despite occlusion of the receptor binding site, the spike induces potently neutralizing sera against multiple SARS-CoV-2 variants. Antibodies are raised against a different pattern of epitopes to those induced by other spike constructs, preferring conformational epitopes present in the closed conformation. Closed spikes, or mRNA vaccines based on their sequence, can be a valuable component of next-generation vaccines.


Assuntos
Enzima de Conversão de Angiotensina 2 , Anticorpos Neutralizantes , Anticorpos Antivirais , Epitopos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/imunologia , Animais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/química , Vacinas contra COVID-19/imunologia , Epitopos/química , Epitopos/imunologia , Células HEK293 , Humanos , Camundongos , Estabilidade Proteica , SARS-CoV-2/química , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia
4.
Eur Biophys J ; 50(3-4): 411-427, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33881594

RESUMO

Microscale thermophoresis (MST), and the closely related Temperature Related Intensity Change (TRIC), are synonyms for a recently developed measurement technique in the field of biophysics to quantify biomolecular interactions, using the (capillary-based) NanoTemper Monolith and (multiwell plate-based) Dianthus instruments. Although this technique has been extensively used within the scientific community due to its low sample consumption, ease of use, and ubiquitous applicability, MST/TRIC has not enjoyed the unambiguous acceptance from biophysicists afforded to other biophysical techniques like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR). This might be attributed to several facts, e.g., that various (not fully understood) effects are contributing to the signal, that the technique is licensed to only a single instrument developer, NanoTemper Technology, and that its reliability and reproducibility have never been tested independently and systematically. Thus, a working group of ARBRE-MOBIEU has set up a benchmark study on MST/TRIC to assess this technique as a method to characterize biomolecular interactions. Here we present the results of this study involving 32 scientific groups within Europe and two groups from the US, carrying out experiments on 40 Monolith instruments, employing a standard operation procedure and centrally prepared samples. A protein-small molecule interaction, a newly developed protein-protein interaction system and a pure dye were used as test systems. We characterized the instrument properties and evaluated instrument performance, reproducibility, the effect of different analysis tools, the influence of the experimenter during data analysis, and thus the overall reliability of this method.


Assuntos
Benchmarking , Laboratórios , Calorimetria , Reprodutibilidade dos Testes , Temperatura
5.
Chem Sci ; 12(3): 880-895, 2021 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-33623657

RESUMO

Here we exploit the simple, ultra-stable, modular architecture of consensus-designed tetratricopeptide repeat proteins (CTPRs) to create a platform capable of displaying both single as well as multiple functions and with diverse programmable geometrical arrangements by grafting non-helical short linear binding motifs (SLiMs) onto the loops between adjacent repeats. As proof of concept, we built synthetic CTPRs to bind and inhibit the human tankyrase proteins (hTNKS), which play a key role in Wnt signaling and are upregulated in cancer. A series of mono-valent and multi-valent hTNKS binders was assembled. To fully exploit the modular scaffold and to further diversify the multi-valent geometry, we engineered the binding modules with two different formats, one monomeric and the other trimeric. We show that the designed proteins are stable, correctly folded and capable of binding to and inhibiting the cellular activity of hTNKS leading to downregulation of the Wnt pathway. Multivalency in both the CTPR protein arrays and the hTNKS target results in the formation of large macromolecular assemblies, which can be visualized both in vitro and in the cell. When delivered into the cell by nanoparticle encapsulation, the multivalent CTPR proteins displayed exceptional activity. They are able to inhibit Wnt signaling where small molecule inhibitors have failed to date. Our results point to the tremendous potential of the CTPR platform to exploit a range of SLiMs and assemble synthetic binding molecules with built-in multivalent capabilities and precise, pre-programmed geometries.

6.
Nat Struct Mol Biol ; 28(3): 278-289, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33633400

RESUMO

Trim-Away is a recently developed technology that exploits off-the-shelf antibodies and the RING E3 ligase and cytosolic antibody receptor TRIM21 to carry out rapid protein depletion. How TRIM21 is catalytically activated upon target engagement, either during its normal immune function or when repurposed for targeted protein degradation, is unknown. Here we show that a mechanism of target-induced clustering triggers intermolecular dimerization of the RING domain to switch on the ubiquitination activity of TRIM21 and induce virus neutralization or drive Trim-Away. We harness this mechanism for selective degradation of disease-causing huntingtin protein containing long polyglutamine tracts and expand the Trim-Away toolbox with highly active TRIM21-nanobody chimeras that can also be controlled optogenetically. This work provides a mechanism for cellular activation of TRIM RING ligases and has implications for targeted protein degradation technologies.


Assuntos
Proteólise , Ribonucleoproteínas/metabolismo , Ubiquitinação , Animais , Biocatálise , Linhagem Celular , Drosophila melanogaster/citologia , Humanos , Proteína Huntingtina/química , Proteína Huntingtina/metabolismo , Camundongos , Modelos Moleculares , Optogenética , Peptídeos/metabolismo , Ligação Proteica , Multimerização Proteica , Ribonucleoproteínas/química , Ubiquitina-Proteína Ligases/metabolismo
7.
Biophys J ; 119(11): 2205-2218, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33137306

RESUMO

VPS34 complex II (VPS34CII) is a 386-kDa assembly of the lipid kinase subunit VPS34 and three regulatory subunits that altogether function as a prototypical class III phosphatidylinositol-3-kinase (PI3K). When the active VPS34CII complex is docked to the cytoplasmic surface of endosomal membranes, it phosphorylates its substrate lipid (phosphatidylinositol, PI) to generate the essential signaling lipid phosphatidylinositol-3-phosphate (PI3P). In turn, PI3P recruits an array of signaling proteins containing PI3P-specific targeting domains (including FYVE, PX, and PROPPINS) to the membrane surface, where they initiate key cell processes. In endocytosis and early endosome development, net VPS34CII-catalyzed PI3P production is greatly amplified by Rab5A, a small G protein of the Ras GTPase superfamily. Moreover, VPS34CII and Rab5A are each strongly linked to multiple human diseases. Thus, a molecular understanding of the mechanism by which Rab5A activates lipid kinase activity will have broad impacts in both signaling biology and medicine. Two general mechanistic models have been proposed for small G protein activation of PI3K lipid kinases. 1) In the membrane recruitment mechanism, G protein association increases the density of active kinase on the membrane. And 2) in the allosteric activation mechanism, G protein allosterically triggers an increase in the specific activity (turnover rate) of the membrane-bound kinase molecule. This study employs an in vitro single-molecule approach to elucidate the mechanism of GTP-Rab5A-associated VPS34CII kinase activation in a reconstituted GTP-Rab5A-VPS34CII-PI3P-PX signaling pathway on a target membrane surface. The findings reveal that both membrane recruitment and allosteric mechanisms make important contributions to the large increase in VPS34CII kinase activity and PI3P production triggered by membrane-anchored GTP-Rab5A. Notably, under near-physiological conditions in the absence of other activators, membrane-anchored GTP-Rab5A provides strong, virtually binary on-off switching and is required for VPS34CII membrane binding and PI3P production.


Assuntos
Classe III de Fosfatidilinositol 3-Quinases , Endossomos , Proteínas rab5 de Ligação ao GTP , Endocitose , Humanos , Membranas Intracelulares , Fosfatidilinositóis
8.
Biochemistry ; 59(43): 4155-4162, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-32818369

RESUMO

Microbial nucleic acids in the extracellular milieu are recognized in vertebrates by Toll-like receptors (TLRs), one of the most important families of innate immune receptors. TLR9 recognizes single-stranded unmethylated CpG DNA in endosomes. DNA binding induces TLR9 dimerization and activation of a potent inflammatory response. To provide insights on how DNA ligands induce TLR9 dimerization, we developed a detailed theoretical framework for equilibrium ligand binding, modeling the binding of the ssDNA at the two main sites on the TLR9 ectodomain. Light scattering and fluorescence anisotropy assays performed with recombinant TLR9 ectodomain and a panel of agonistic and antagonistic DNA ligands provide data that restrain the binding parameters, identify the likely ligand binding intermediates, and suggest cooperative modes of binding. This work brings us one step closer to establishing a rigorous biochemical understanding of how TLRs are activated by their ligands.


Assuntos
Receptor Toll-Like 9/química , Receptor Toll-Like 9/metabolismo , Animais , Anisotropia , Sítios de Ligação , Ilhas de CpG/fisiologia , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Difusão Dinâmica da Luz , Polarização de Fluorescência , Humanos , Hidrodinâmica , Camundongos , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/metabolismo
9.
Nat Commun ; 11(1): 3464, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651375

RESUMO

DOCK (dedicator of cytokinesis) proteins are multidomain guanine nucleotide exchange factors (GEFs) for RHO GTPases that regulate intracellular actin dynamics. DOCK proteins share catalytic (DOCKDHR2) and membrane-associated (DOCKDHR1) domains. The structurally-related DOCK1 and DOCK2 GEFs are specific for RAC, and require ELMO (engulfment and cell motility) proteins for function. The N-terminal RAS-binding domain (RBD) of ELMO (ELMORBD) interacts with RHOG to modulate DOCK1/2 activity. Here, we determine the cryo-EM structures of DOCK2-ELMO1 alone, and as a ternary complex with RAC1, together with the crystal structure of a RHOG-ELMO2RBD complex. The binary DOCK2-ELMO1 complex adopts a closed, auto-inhibited conformation. Relief of auto-inhibition to an active, open state, due to a conformational change of the ELMO1 subunit, exposes binding sites for RAC1 on DOCK2DHR2, and RHOG and BAI GPCRs on ELMO1. Our structure explains how up-stream effectors, including DOCK2 and ELMO1 phosphorylation, destabilise the auto-inhibited state to promote an active GEF.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Calorimetria , Proteínas Ativadoras de GTPase/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Cinética , Microscopia Eletrônica , Fosforilação , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
10.
Structure ; 28(6): 674-689.e11, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32375023

RESUMO

Centrioles are cylindrical assemblies whose peripheral microtubule array displays a 9-fold rotational symmetry that is established by the scaffolding protein SAS6. Centriole symmetry can be broken by centriole-associated structures, such as the striated fibers in Chlamydomonas that are important for ciliary function. The conserved protein CCDC61/VFL3 is involved in this process, but its exact role is unclear. Here, we show that CCDC61 is a paralog of SAS6. Crystal structures of CCDC61 demonstrate that it contains two homodimerization interfaces that are similar to those found in SAS6, but result in the formation of linear filaments rather than rings. Furthermore, we show that CCDC61 binds microtubules and that residues involved in CCDC61 microtubule binding are important for ciliary function in Chlamydomonas. Together, our findings suggest that CCDC61 and SAS6 functionally diverged from a common ancestor while retaining the ability to scaffold the assembly of basal body-associated structures or centrioles, respectively.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Chlamydomonas/fisiologia , Cílios/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Algas/química , Proteínas de Algas/metabolismo , Linhagem Celular , Chlamydomonas/classificação , Cristalografia por Raios X , Células HEK293 , Humanos , Microtúbulos/metabolismo , Modelos Moleculares , Filogenia , Conformação Proteica , Domínios Proteicos , Multimerização Proteica
11.
Nat Commun ; 10(1): 4502, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31582740

RESUMO

The cytosolic antibody receptor TRIM21 possesses unique ubiquitination activity that drives broad-spectrum anti-pathogen targeting and underpins the protein depletion technology Trim-Away. This activity is dependent on formation of self-anchored, K63-linked ubiquitin chains by the heterodimeric E2 enzyme Ube2N/Ube2V2. Here we reveal how TRIM21 facilitates ubiquitin transfer and differentiates this E2 from other closely related enzymes. A tri-ionic motif provides optimally distributed anchor points that allow TRIM21 to wrap an Ube2N~Ub complex around its RING domain, locking the closed conformation and promoting ubiquitin discharge. Mutation of these anchor points inhibits ubiquitination with Ube2N/Ube2V2, viral neutralization and immune signalling. We show that the same mechanism is employed by the anti-HIV restriction factor TRIM5 and identify spatially conserved ionic anchor points in other Ube2N-recruiting RING E3s. The tri-ionic motif is exclusively required for Ube2N but not Ube2D1 activity and provides a generic E2-specific catalysis mechanism for RING E3s.


Assuntos
Lisina/metabolismo , Ribonucleoproteínas/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação/fisiologia , Motivos de Aminoácidos/genética , Biocatálise , Cristalografia por Raios X , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Mutação , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica/genética , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Ubiquitina-Proteína Ligases/metabolismo
12.
Nature ; 574(7777): 278-282, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31578520

RESUMO

In eukaryotes, accurate chromosome segregation in mitosis and meiosis maintains genome stability and prevents aneuploidy. Kinetochores are large protein complexes that, by assembling onto specialized Cenp-A nucleosomes1,2, function to connect centromeric chromatin to microtubules of the mitotic spindle3,4. Whereas the centromeres of vertebrate chromosomes comprise millions of DNA base pairs and attach to multiple microtubules, the simple point centromeres of budding yeast are connected to individual microtubules5,6. All 16 budding yeast chromosomes assemble complete kinetochores using a single Cenp-A nucleosome (Cenp-ANuc), each of which is perfectly centred on its cognate centromere7-9. The inner and outer kinetochore modules are responsible for interacting with centromeric chromatin and microtubules, respectively. Here we describe the cryo-electron microscopy structure of the Saccharomyces cerevisiae inner kinetochore module, the constitutive centromere associated network (CCAN) complex, assembled onto a Cenp-A nucleosome (CCAN-Cenp-ANuc). The structure explains the interdependency of the constituent subcomplexes of CCAN and shows how the Y-shaped opening of CCAN accommodates Cenp-ANuc to enable specific CCAN subunits to contact the nucleosomal DNA and histone subunits. Interactions with the unwrapped DNA duplex at the two termini of Cenp-ANuc are mediated predominantly by a DNA-binding groove in the Cenp-L-Cenp-N subcomplex. Disruption of these interactions impairs assembly of CCAN onto Cenp-ANuc. Our data indicate a mechanism of Cenp-A nucleosome recognition by CCAN and how CCAN acts as a platform for assembly of the outer kinetochore to link centromeres to the mitotic spindle for chromosome segregation.


Assuntos
Proteína Centromérica A/metabolismo , Cinetocoros/química , Cinetocoros/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Proteína Centromérica A/química , Proteína Centromérica A/ultraestrutura , Microscopia Crioeletrônica , DNA/química , DNA/metabolismo , DNA/ultraestrutura , Cinetocoros/ultraestrutura , Modelos Moleculares , Complexos Multiproteicos/ultraestrutura , Nucleossomos/ultraestrutura , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura
13.
Nat Microbiol ; 4(12): 2357-2368, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31501539

RESUMO

Bactofilins are small ß-helical proteins that form cytoskeletal filaments in a range of bacteria. Bactofilins have diverse functions, from cell stalk formation in Caulobacter crescentus to chromosome segregation and motility in Myxococcus xanthus. However, the precise molecular architecture of bactofilin filaments has remained unclear. Here, sequence analysis and electron microscopy results reveal that, in addition to being widely distributed across bacteria and archaea, bactofilins are also present in a few eukaryotic lineages such as the Oomycetes. Electron cryomicroscopy analysis demonstrated that the sole bactofilin from Thermus thermophilus (TtBac) forms constitutive filaments that polymerize through end-to-end association of the ß-helical domains. Using a nanobody, we determined the near-atomic filament structure, showing that the filaments are non-polar. A polymerization-impairing mutation enabled crystallization and structure determination, while reaffirming the lack of polarity and the strength of the ß-stacking interface. To confirm the generality of the lack of polarity, we performed coevolutionary analysis on a large set of sequences. Finally, we determined that the widely conserved N-terminal disordered tail of TtBac is responsible for direct binding to lipid membranes, both on liposomes and in Escherichia coli cells. Membrane binding is probably a common feature of these widespread but only recently discovered filaments of the prokaryotic cytoskeleton.


Assuntos
Archaea/citologia , Bactérias/citologia , Citoesqueleto/química , Citoesqueleto/ultraestrutura , Sequência de Aminoácidos , Archaea/química , Bactérias/química , Proteínas de Bactérias/química , Caulobacter crescentus/química , Caulobacter crescentus/citologia , Segregação de Cromossomos , Microscopia Crioeletrônica , Proteínas do Citoesqueleto/química , Escherichia coli , Lipossomos , Membranas , Modelos Moleculares , Myxococcus xanthus , Análise de Sequência
14.
EMBO J ; 38(21): e102177, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31531998

RESUMO

AMPylation is an inactivating modification that alters the activity of the major endoplasmic reticulum (ER) chaperone BiP to match the burden of unfolded proteins. A single ER-localised Fic protein, FICD (HYPE), catalyses both AMPylation and deAMPylation of BiP. However, the basis for the switch in FICD's activity is unknown. We report on the transition of FICD from a dimeric enzyme, that deAMPylates BiP, to a monomer with potent AMPylation activity. Mutations in the dimer interface, or of residues along an inhibitory pathway linking the dimer interface to the enzyme's active site, favour BiP AMPylation in vitro and in cells. Mechanistically, monomerisation relieves a repressive effect allosterically propagated from the dimer interface to the inhibitory Glu234, thereby permitting AMPylation-competent binding of MgATP. Moreover, a reciprocal signal, propagated from the nucleotide-binding site, provides a mechanism for coupling the oligomeric state and enzymatic activity of FICD to the energy status of the ER.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Células HEK293 , Humanos , Conformação Proteica
15.
Proc Natl Acad Sci U S A ; 116(30): 15042-15051, 2019 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-31289231

RESUMO

Transcription of transposable elements is tightly regulated to prevent genome damage. KRAB domain-containing zinc finger proteins (KRAB-ZFPs) and KRAB-associated protein 1 (KAP1/TRIM28) play a key role in regulating retrotransposons. KRAB-ZFPs recognize specific retrotransposon sequences and recruit KAP1, inducing the assembly of an epigenetic silencing complex, with chromatin remodeling activities that repress transcription of the targeted retrotransposon and adjacent genes. Our biophysical and structural data show that the tripartite motif (TRIM) of KAP1 forms antiparallel dimers, which further assemble into tetramers and higher-order oligomers in a concentration-dependent manner. Structure-based mutations in the B-box 1 domain prevent higher-order oligomerization without significant loss of retrotransposon silencing activity, indicating that, in contrast to other TRIM-family proteins, self-assembly is not essential for KAP1 function. The crystal structure of the KAP1 TRIM dimer identifies the KRAB domain binding site in the coiled-coil domain near the dyad. Mutations at this site abolished KRAB binding and transcriptional silencing activity of KAP1. This work identifies the interaction interfaces in the KAP1 TRIM responsible for self-association and KRAB binding and establishes their role in retrotransposon silencing.


Assuntos
Epigênese Genética , Inativação Gênica , Proteínas Repressoras/química , Retroelementos , Proteína 28 com Motivo Tripartido/química , Sequência de Aminoácidos , Sítios de Ligação , Cromatina/química , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Genética , Proteína 28 com Motivo Tripartido/genética , Proteína 28 com Motivo Tripartido/metabolismo
16.
Proc Natl Acad Sci U S A ; 116(11): 4946-4954, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30804176

RESUMO

Cells dynamically adjust their protein translation profile to maintain homeostasis in changing environments. During nutrient stress, the kinase general control nonderepressible 2 (GCN2) phosphorylates translation initiation factor eIF2α, initiating the integrated stress response (ISR). To examine the mechanism of GCN2 activation, we have reconstituted this process in vitro, using purified components. We find that recombinant human GCN2 is potently stimulated by ribosomes and, to a lesser extent, by tRNA. Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) mapped GCN2-ribosome interactions to domain II of the uL10 subunit of the ribosomal P-stalk. Using recombinant, purified P-stalk, we showed that this domain of uL10 is the principal component of binding to GCN2; however, the conserved 14-residue C-terminal tails (CTTs) in the P1 and P2 P-stalk proteins are also essential for GCN2 activation. The HisRS-like and kinase domains of GCN2 show conformational changes upon binding recombinant P-stalk complex. Given that the ribosomal P-stalk stimulates the GTPase activity of elongation factors during translation, we propose that the P-stalk could link GCN2 activation to translational stress, leading to initiation of ISR.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Ribossomos/metabolismo , Motivos de Aminoácidos , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Fosforilação , Domínios Proteicos , Proteínas Serina-Treonina Quinases/química , Relação Estrutura-Atividade
17.
Nat Struct Mol Biol ; 25(12): 1103-1110, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30478265

RESUMO

Kinetochores are multicomponent complexes responsible for coordinating the attachment of centromeric DNA to mitotic-spindle microtubules. The point centromeres of budding yeast are organized into three centromeric determining elements (CDEs), and are associated with the centromere-specific nucleosome Cse4. Deposition of Cse4 at CEN loci is dependent on the CBF3 complex that engages CDEIII to direct Cse4 nucleosomes to CDEII. To understand how CBF3 recognizes CDEIII and positions Cse4, we determined a cryo-EM structure of a CBF3-CEN complex. CBF3 interacts with CEN DNA as a head-to-head dimer that includes the whole of CDEIII and immediate 3' regions. Specific CEN-binding of CBF3 is mediated by a Cep3 subunit of one of the CBF3 protomers that forms major groove interactions with the conserved and essential CCG and TGT motifs of CDEIII. We propose a model for a CBF3-Cse4-CEN complex with implications for understanding CBF3-directed deposition of the Cse4 nucleosome at CEN loci.


Assuntos
Proteínas de Ligação a DNA/química , Cinetocoros/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Modelos Moleculares , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia
18.
mBio ; 9(5)2018 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-30206170

RESUMO

Most bacteria and archaea use the tubulin homologue FtsZ as its central organizer of cell division. In Gram-negative Escherichia coli bacteria, FtsZ recruits cytosolic, transmembrane, periplasmic, and outer membrane proteins, assembling the divisome that facilitates bacterial cell division. One such divisome component, FtsQ, a bitopic membrane protein with a globular domain in the periplasm, has been shown to interact with many other divisome proteins. Despite its otherwise unknown function, it has been shown to be a major divisome interaction hub. Here, we investigated the interactions of FtsQ with FtsB and FtsL, two small bitopic membrane proteins that act immediately downstream of FtsQ. We show in biochemical assays that the periplasmic domains of E. coli FtsB and FtsL interact with FtsQ, but not with each other. Our crystal structure of FtsB bound to the ß domain of FtsQ shows that only residues 64 to 87 of FtsB interact with FtsQ. A synthetic peptide comprising those 24 FtsB residues recapitulates the FtsQ-FtsB interactions. Protein deletions and structure-guided mutant analyses validate the structure. Furthermore, the same structure-guided mutants show cell division defects in vivo that are consistent with our structure of the FtsQ-FtsB complex that shows their interactions as they occur during cell division. Our work provides intricate details of the interactions within the divisome and also provides a tantalizing view of a highly conserved protein interaction in the periplasm of bacteria that is an excellent target for cell division inhibitor searches.IMPORTANCE In most bacteria and archaea, filaments of FtsZ protein organize cell division. FtsZ forms a ring structure at the division site and starts the recruitment of 10 to 20 downstream proteins that together form a multiprotein complex termed the divisome. The divisome is thought to facilitate many of the steps required to make two cells out of one. FtsQ and FtsB are part of the divisome, with FtsQ being a central hub, interacting with most of the other divisome components. Here we show for the first time in detail how FtsQ interacts with its downstream partner FtsB and show that mutations that disturb the interface between the two proteins effectively inhibit cell division.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Ciclo Celular/genética , Cristalografia por Raios X , Análise Mutacional de DNA , Proteínas de Escherichia coli/genética , Deleção de Genes , Proteínas de Membrana/genética , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas
19.
Elife ; 72018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29944118

RESUMO

Polarised mRNA transport is a prevalent mechanism for spatial control of protein synthesis. However, the composition of transported ribonucleoprotein particles (RNPs) and the regulation of their movement are poorly understood. We have reconstituted microtubule minus end-directed transport of mRNAs using purified components. A Bicaudal-D (BicD) adaptor protein and the RNA-binding protein Egalitarian (Egl) are sufficient for long-distance mRNA transport by the dynein motor and its accessory complex dynactin, thus defining a minimal transport-competent RNP. Unexpectedly, the RNA is required for robust activation of dynein motility. We show that a cis-acting RNA localisation signal promotes the interaction of Egl with BicD, which licenses the latter protein to recruit dynein and dynactin. Our data support a model for BicD activation based on RNA-induced occupancy of two Egl-binding sites on the BicD dimer. Scaffolding of adaptor protein assemblies by cargoes is an attractive mechanism for regulating intracellular transport.


Assuntos
Proteínas de Drosophila/genética , Complexo Dinactina/genética , Dineínas/genética , Animais , Sítios de Ligação , Dineínas do Citoplasma/química , Dineínas do Citoplasma/genética , Proteínas de Drosophila/química , Drosophila melanogaster/genética , Complexo Dinactina/química , Dineínas/química , Ligação Proteica/genética , Multimerização Proteica , Transporte Proteico/genética , Transporte de RNA/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genética
20.
Nat Commun ; 9(1): 1731, 2018 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-29712910

RESUMO

Centrosomes are required for faithful chromosome segregation during mitosis. They are composed of a centriole pair that recruits and organizes the microtubule-nucleating pericentriolar material. Centriole duplication is tightly controlled in vivo and aberrations in this process are associated with several human diseases, including cancer and microcephaly. Although factors essential for centriole assembly, such as STIL and PLK4, have been identified, the underlying molecular mechanisms that drive this process are incompletely understood. Combining protein proximity mapping with high-resolution structural methods, we identify CEP85 as a centriole duplication factor that directly interacts with STIL through a highly conserved interaction interface involving a previously uncharacterised domain of STIL. Structure-guided mutational analyses in vivo demonstrate that this interaction is essential for efficient centriolar targeting of STIL, PLK4 activation and faithful daughter centriole assembly. Taken together, our results illuminate a molecular mechanism underpinning the spatiotemporal regulation of the early stages of centriole duplication.


Assuntos
Centríolos/metabolismo , Segregação de Cromossomos , Proteínas do Citoesqueleto/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas de Fusão Oncogênica/química , Proteínas Serina-Treonina Quinases/química , Sítios de Ligação , Linhagem Celular Tumoral , Centríolos/ultraestrutura , Cristalografia por Raios X , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitose , Modelos Moleculares , Mutação , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
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