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1.
Nat Commun ; 10(1): 2641, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201325

RESUMO

Epsilon toxin (Etx), a potent pore forming toxin (PFT) produced by Clostridium perfringens, is responsible for the pathogenesis of enterotoxaemia of ruminants and has been suggested to play a role in multiple sclerosis in humans. Etx is a member of the aerolysin family of ß-PFTs (aß-PFTs). While the Etx soluble monomer structure was solved in 2004, Etx pore structure has remained elusive due to the difficulty of isolating the pore complex. Here we show the cryo-electron microscopy structure of Etx pore assembled on the membrane of susceptible cells. The pore structure explains important mutant phenotypes and suggests that the double ß-barrel, a common feature of the aß-PFTs, may be an important structural element in driving efficient pore formation. These insights provide the framework for the development of novel therapeutics to prevent human and animal infections, and are relevant for nano-biotechnology applications.


Assuntos
Toxinas Bacterianas/química , Clostridium perfringens/ultraestrutura , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/metabolismo , Biotecnologia/métodos , Linhagem Celular , Infecções por Clostridium/microbiologia , Infecções por Clostridium/prevenção & controle , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Clostridium perfringens/patogenicidade , Microscopia Crioeletrônica , Cães , Enterotoxemia/microbiologia , Enterotoxemia/prevenção & controle , Modelos Moleculares , Mutagênese Sítio-Dirigida , Nanotecnologia/métodos , Conformação Proteica em Folha beta/genética , Multimerização Proteica/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
2.
J Mol Biol ; 430(18 Pt B): 3297-3310, 2018 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-29969581

RESUMO

Heterogeneity in small heat shock proteins (sHsps) spans multiple spatiotemporal regimes-from fast fluctuations of part of the protein, to conformational variability of tertiary structure, plasticity of the interfaces, and polydispersity of the inter-converting, and co-assembling oligomers. This heterogeneity and dynamic nature of sHsps has significantly hindered their structural characterization. Atomic coordinates are particularly lacking for vertebrate sHsps, where most available structures are of extensively truncated homomers. sHsps play important roles in maintaining protein levels in the cell and therefore in organismal health and disease. HspB2 and HspB3 are vertebrate sHsps that are found co-assembled in neuromuscular cells, and variants thereof are associated with disease. Here, we present the structure of human HspB2/B3, which crystallized as a hetero-tetramer in a 3:1 ratio. In the HspB2/B3 tetramer, the four α-crystallin domains (ACDs) assemble into a flattened tetrahedron which is pierced by two non-intersecting approximate dyads. Assembly is mediated by flexible "nuts and bolts" involving IXI/V motifs from terminal regions filling ACD pockets. Parts of the N-terminal region bind in an unfolded conformation into the anti-parallel shared ACD dimer grooves. Tracts of the terminal regions are not resolved, most likely due to their disorder in the crystal lattice. This first structure of a full-length human sHsp heteromer reveals the heterogeneous interactions of the terminal regions and suggests a plasticity that is important for the cytoprotective functions of sHsps.

4.
Nucleic Acids Res ; 46(D1): D486-D492, 2018 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-29126160

RESUMO

The Protein Data Bank in Europe (PDBe, pdbe.org) is actively engaged in the deposition, annotation, remediation, enrichment and dissemination of macromolecular structure data. This paper describes new developments and improvements at PDBe addressing three challenging areas: data enrichment, data dissemination and functional reusability. New features of the PDBe Web site are discussed, including a context dependent menu providing links to raw experimental data and improved presentation of structures solved by hybrid methods. The paper also summarizes the features of the LiteMol suite, which is a set of services enabling fast and interactive 3D visualization of structures, with associated experimental maps, annotations and quality assessment information. We introduce a library of Web components which can be easily reused to port data and functionality available at PDBe to other services. We also introduce updates to the SIFTS resource which maps PDB data to other bioinformatics resources, and the PDBe REST API.

5.
Biochim Biophys Acta ; 1860(1 Pt B): 304-14, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26145577

RESUMO

BACKGROUND: Lens transparency is due to the ordered arrangement of the major structural proteins, called crystallins. ßB2 crystallin in the lens of the eye readily forms dimers with other ß-crystallin subunits, but the resulting heterodimer structures are not known and were investigated in this study. METHODS: Structures of ßA3 and ßB2 crystallin homodimers and the ßA3/ßB2 crystallin heterodimers were probed by measuring changes in solvent accessibility using hydrogen-deuterium exchange with mass spectrometry. We further mimicked deamidation in ßB2 and probed the effect on the ßA3/ßB2 heterodimer. Results were confirmed with chemical crosslinking and NMR. RESULTS: Both ßA3 and ßB2 had significantly decreased deuterium levels in the heterodimer compared to their respective homodimers, suggesting that they had less solvent accessibility and were more compact in the heterodimer. The compact structure of ßB2 was supported by the identification of chemical crosslinks between lysines in ßB2 within the heterodimer that were inconsistent with ßB2's extended homodimeric structure. The compact structure of ßA3 was supported by an overall decrease in mobility of ßA3 in the heterodimer detected by NMR. In ßB2, peptides 70-84 and 121-134 were exposed in the homodimer, but buried in the heterodimer with ≥50% decreases in deuterium levels. Homologous peptides in ßA3, 97-109 and 134-149, had 25-50% decreases in deuterium levels in the heterodimer. These peptides are probable sites of interaction between ßB2 and ßA3 and are located at the predicted interface between subunits with bent linkers. Deamidation at Q184 in ßB2 at this predicted interface led to a less compact ßB2 in the heterodimer. The more compact structure of the ßA3/ßB2 heterodimer was also more heat stable than either of the homodimers. CONCLUSIONS: The major structural proteins in the lens, the ß-crystallins, are not static, but dynamic in solution, with differences in accessibility between the homo-and hetero-dimers. This structural flexibility, particularly of ßB2, may facilitate formation of different size higher-ordered structures found in the transparent lens. GENERAL SIGNIFICANCE: Understanding complex hetero-oligomer interactions between ß-crystallins in normal lens and how these interactions change during aging is fundamental to understanding the cause of cataracts. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.


Assuntos
Amidas/química , Medição da Troca de Deutério/métodos , Cristalino/química , Multimerização Proteica , beta-Cristalinas/química , beta-Cristalinas/ultraestrutura , Sequência de Aminoácidos , Animais , Sítios de Ligação , Dimerização , Humanos , Técnicas de Sonda Molecular , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica
6.
Nucleic Acids Res ; 44(D1): D385-95, 2016 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-26476444

RESUMO

The Protein Data Bank in Europe (http://pdbe.org) accepts and annotates depositions of macromolecular structure data in the PDB and EMDB archives and enriches, integrates and disseminates structural information in a variety of ways. The PDBe website has been redesigned based on an analysis of user requirements, and now offers intuitive access to improved and value-added macromolecular structure information. Unique value-added information includes lists of reviews and research articles that cite or mention PDB entries as well as access to figures and legends from full-text open-access publications that describe PDB entries. A powerful new query system not only shows all the PDB entries that match a given query, but also shows the 'best structures' for a given macromolecule, ligand complex or sequence family using data-quality information from the wwPDB validation reports. A PDBe RESTful API has been developed to provide unified access to macromolecular structure data available in the PDB and EMDB archives as well as value-added annotations, e.g. regarding structure quality and up-to-date cross-reference information from the SIFTS resource. Taken together, these new developments facilitate unified access to macromolecular structure data in an intuitive way for non-expert users and support expert users in analysing macromolecular structure data.


Assuntos
Bases de Dados de Proteínas , Conformação Proteica , Internet , Microscopia Eletrônica , Modelos Moleculares , Interface Usuário-Computador
7.
J Mol Med (Berl) ; 93(7): 773-82, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25686753

RESUMO

UNLABELLED: Filamin A, the filamentous protein encoded by the X-linked gene FLNA, cross-links cytoskeletal actin into three-dimensional networks, facilitating its role as a signalling scaffold and a mechanosensor of extrinsic shear forces. Central to these functions is the ability of FLNA to form V-shaped homodimers through its C-terminal located filamin repeat 24. Additionally, many proteins that interact with FLNA have a binding site that includes the C-terminus of the protein. Here, a cohort of patients with mutations affecting this region of the protein is studied, with particular emphasis on the phenotype of male hemizygotes. Seven unrelated families are reported, with five exhibiting a typical female presentation of periventricular heterotopia (PH), a neuronal migration disorder typically caused by loss-of-function mutations in FLNA. One male presents with widespread PH consistent with previous male phenotypes attributable to hypomorphic mutations in FLNA. In stark contrast, two brothers are described with a mild PH presentation, due to a missense mutation (p.Gly2593Glu) inserting a large negatively charged amino acid into the hydrophobic dimerisation interface of FLNA. Co-immunoprecipitation, in vitro cross-linking studies and gel filtration chromatography all demonstrated that homodimerisation of isolated FLNA repeat 24 is abolished by this p.Gly2593Glu substitution but that extended FLNA(Gly2593Glu) repeat 16-24 constructs exhibit dimerisation. These observations imply that other interactions apart from those mediated by the canonical repeat 24 dimerisation interface contribute to FLNA homodimerisation and that mutations affecting this region of the protein can have broad phenotypic effects. KEY MESSAGES: • Mutations in the X-linked gene FLNA cause a spectrum of syndromes. • Genotype-phenotype correlations are emerging but still remain unclear. • C-term mutations can confer male lethality, survival or connective tissue defects. • Mutations leading to the latter affect filamin dimerisation. • This deficit is compensated for by remotely acting domains elsewhere in FLNA.


Assuntos
Filaminas/genética , Heterotopia Nodular Periventricular/genética , Multimerização Proteica/genética , Sequência de Aminoácidos , Movimento Celular/genética , Feminino , Fibroblastos , Estudos de Associação Genética , Humanos , Masculino , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , Fenótipo , Estrutura Terciária de Proteína
8.
Structure ; 21(2): 193-4, 2013 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-23394940

RESUMO

A crystal structure of a yeast small heat shock protein reported by Hanazono and colleagues in this issue of Structure reveals the versatility of the α-crystallin domain dimer for building assemblies of different size and symmetry. The domains assemble into a vessel filled with hydrophobic sequence extensions enriched with phenylalanines.


Assuntos
Proteínas de Choque Térmico/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces
9.
Protein Sci ; 22(4): 367-80, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23389822

RESUMO

The camera eye lens of vertebrates is a classic example of the re-engineering of existing protein components to fashion a new device. The bulk of the lens is formed from proteins belonging to two superfamilies, the α-crystallins and the ßγ-crystallins. Tracing their ancestry may throw light on the origin of the optics of the lens. The α-crystallins belong to the ubiquitous small heat shock proteins family that plays a protective role in cellular homeostasis. They form enormous polydisperse oligomers that challenge modern biophysical methods to uncover the molecular basis of their assembly structure and chaperone-like protein binding function. It is argued that a molecular phenotype of a dynamic assembly suits a chaperone function as well as a structural role in the eye lens where the constraint of preventing protein condensation is paramount. The main cellular partners of α-crystallins, the ß- and γ-crystallins, have largely been lost from the animal kingdom but the superfamily is hugely expanded in the vertebrate eye lens. Their structures show how a simple Greek key motif can evolve rapidly to form a complex array of monomers and oligomers. Apart from remaining transparent, a major role of the partnership of α-crystallins with ß- and γ-crystallins in the lens is to form a refractive index gradient. Here, we show some of the structural and genetic features of these two protein superfamilies that enable the rapid creation of different assembly states, to match the rapidly changing optical needs among the various vertebrates.


Assuntos
Cristalinas/química , Cristalinas/fisiologia , Cristalino/química , Cristalino/fisiologia , Chaperonas Moleculares/química , Chaperonas Moleculares/fisiologia , Animais , Cristalinas/genética , Peixes , Humanos , Camundongos , Modelos Moleculares , Chaperonas Moleculares/genética , Refratometria
10.
Chem Biol ; 19(5): 547-8, 2012 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-22633405
11.
Int J Biochem Cell Biol ; 44(10): 1687-97, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22405853

RESUMO

α-Crystallin, a major component of the eye lens cytoplasm, is a large multimer formed from two members of the small heat shock protein (sHsp) family. Inherited crystallin mutations are a common cause of childhood cataract, whereas miscellaneous changes to the long-lived crystallins cause age-related cataract, the most common cause of blindness worldwide. Newly formed eye lens cells use proteostasis to deal with the consequences of mutations, whereas mature lens cells, devoid of the ATP-driven folding and degradation machines, are hypothesized to have the α-crystallin "holdase" chaperone function to prevent protein aggregation. We discuss the impact of truncating and missense mutations on α-crystallin, based on recent progress towards determining sHsp 3D structure. Dominant missense mutations to the "α-crystallin domain" of αA- (HSPB4) or αB-crystallin (HSPB5) occur on residues predicted to facilitate domain dynamics. αB-Crystallin is also expressed in striated muscle and mutations cause myopathy. The impact on these cellular cytoplasms is compared where sHsp multimer partners and metabolic constraints are different. Selected inherited mutations of the lens ß- and γ-crystallins are considered in the context of their possible dependence on the "holdase" chaperone function of α-crystallin. Looking at discrete changes to specific crystallin polypeptide chains that can function as chaperone or substrate provide insights into the workings of a cytoplasmic proteostatic system. These observations provide a framework for validating the function of α-crystallin as a chaperone, or as a lens space filler adapted from a chaperone function. Understanding the mechanistic role of α-crystallins will aid progress in research into age-related cataract and adult-onset myopathy. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.


Assuntos
Catarata/genética , Cristalinas/genética , Proteínas de Choque Térmico Pequenas/genética , Deficiências na Proteostase/genética , Animais , Sítios de Ligação , Catarata/metabolismo , Cristalinas/metabolismo , Proteínas de Choque Térmico Pequenas/metabolismo , Humanos , Cristalino/embriologia , Cristalino/metabolismo , Cristalino/fisiopatologia , Mutação , Conformação Proteica , Deficiências na Proteostase/metabolismo , Estresse Fisiológico
12.
Hum Mol Genet ; 18(24): 4791-800, 2009 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-19773341

RESUMO

Filamin A (FLNA) crosslinks F-actin and binds proteins consistent with roles integrating cell signalling and the cytoskeleton. FLNA missense mutations are associated with the otopalatodigital syndrome (OPD) spectrum of skeletal disorders, clustering in discrete domains. One cluster is found in the second calponin homology domain of the FLNA actin-binding domain (ABD), implicating this region as essential for mediating correct function. Here we show that OPD (FLNA E254K) fibroblast lysates have equivalent concentrations of FLNA compared with controls and that recombinant FLNA E254K ABD has increased in vitro F-actin binding (K(d) 13 microm) compared with wild type (WT; K(d) 48 microm). These observations are consistent with a gain-of-function mechanism for OPD. We have determined the crystal structures of the WT and E254K FLNA ABDs at 2.3 A resolution, revealing that they adopt similar closed conformations. The E254K mutation removes a conserved salt bridge but does not disrupt the ABD structure. The solution structures are also equivalent as determined by circular dichroism spectroscopy, but differential scanning fluorimetry denaturation showed reduced stability (decreased T(m) of 5.6 degrees C) for E254K relative to WT. Ex vivo characterization of E254K OPD patient fibroblasts revealed they have similar motility and adhesion as control cells, implying that many core functions mediated by FLNA are unaffected, consistent with OPD only affecting specific tissues despite FLNA being widely expressed. These data provide the first biochemical evidence for a gain-of-function mechanism for the OPD disorders, and mechanistically distinguishes them from the loss-of-function phenotypes that manifest as disorders of neuronal migration.


Assuntos
Doenças do Desenvolvimento Ósseo/genética , Doenças do Desenvolvimento Ósseo/metabolismo , Movimento Celular/genética , Proteínas Contráteis/genética , Proteínas Contráteis/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Sequência de Aminoácidos , Adesão Celular/genética , Proteínas Contráteis/química , Cristalografia por Raios X , Ossículos da Orelha/anormalidades , Feminino , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Filaminas , Dedos/anormalidades , Humanos , Masculino , Proteínas dos Microfilamentos/química , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/metabolismo , Palato/anormalidades , Estrutura Terciária de Proteína , Síndrome , Dedos do Pé/anormalidades
13.
J Mol Biol ; 390(5): 1030-47, 2009 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-19505475

RESUMO

Missense mutations in filamin B (FLNB) are associated with the autosomal dominant atelosteogenesis (AO) and the Larsen group of skeletal malformation disorders. These mutations cluster in particular FLNB protein domains and act in a presumptive gain-of-function mechanism. In contrast the loss-of-function disorder, spondylocarpotarsal synostosis syndrome, is characterised by the complete absence of FLNB. One cluster of AO missense mutations is found within the second of two calponin homology (CH) domains that create a functional actin-binding domain (ABD). This N-terminal ABD is required for filamin F-actin crosslinking activity, a crucial aspect of filamin's role of integrating cell-signalling events with cellular scaffolding and mechanoprotection. This study characterises the wild type FLNB ABD and investigates the effects of two disease-associated mutations on the structure and function of the FLNB ABD that could explain a gain-of-function mechanism for the AO diseases. We have determined high-resolution X-ray crystal structures of the human filamin B wild type ABD, plus W148R and M202V mutants. All three structures display the classic compact monomeric conformation for the ABD with the CH1 and CH2 domains in close contact. The conservation of tertiary structure in the presence of these mutations shows that the compact ABD conformation is stable to the sequence substitutions. In solution the mutant ABDs display reduced melting temperatures (by 6-7 degrees C) as determined by differential scanning fluorimetry. Characterisation of the wild type and mutant ABD F-actin binding activities via co-sedimentation assays shows that the mutant FLNB ABDs have increased F-actin binding affinities, with dissociation constants of 2.0 microM (W148R) and 0.56 microM (M202V), compared to the wild type ABD K(d) of 7.0 microM. The increased F-actin binding affinity of the mutants presents a biochemical mechanism that differentiates the autosomal dominant gain-of-function FLNB disorders from those that arise through the complete loss of FLNB protein.


Assuntos
Actinas/metabolismo , Substituição de Aminoácidos/genética , Proteínas Contráteis/química , Proteínas Contráteis/genética , Doença/genética , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Sequência de Aminoácidos , Bioensaio , Calmodulina/metabolismo , Proteínas Contráteis/metabolismo , Cristalografia por Raios X , Filaminas , Fluorometria , Humanos , Proteínas dos Microfilamentos/metabolismo , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação/genética , Ligação Proteica , Desnaturação Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Alinhamento de Sequência , Homologia Estrutural de Proteína , Temperatura Ambiente
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