RESUMO
Membraneless organelles (MLOs) are vital and dynamic reaction centers in cells that compartmentalize the cytoplasm in the absence of a membrane. Multivalent interactions between protein low-complexity domains contribute to MLO organization. Previously, we used computational methods to identify structural motifs termed low-complexity amyloid-like reversible kinked segments (LARKS) that promote phase transition to form hydrogels and that are common in human proteins that participate in MLOs. Here, we searched for LARKS in the proteomes of six model organisms: Homo sapiens, Drosophila melanogaster, Plasmodium falciparum, Saccharomyces cerevisiae, Mycobacterium tuberculosis, and Escherichia coli to gain an understanding of the distribution of LARKS in the proteomes of various species. We found that LARKS are abundant in M. tuberculosis, D. melanogaster, and H. sapiens but not in S. cerevisiae or P. falciparum. LARKS have high glycine content, which enables kinks to form as exemplified by the known LARKS-rich amyloidogenic structures of TDP43, FUS, and hnRNPA2, three proteins that are known to participate in MLOs. These results support the idea of LARKS as an evolved structural motif. Based on these results, we also established the LARKSdb Web server, which permits users to search for LARKS in their protein sequences of interest.
Assuntos
Amiloide/química , Proteínas de Drosophila/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Motivos de Aminoácidos , Amiloide/genética , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster , Humanos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The Rosetta software for macromolecular modeling, docking and design is extensively used in laboratories worldwide. During two decades of development by a community of laboratories at more than 60 institutions, Rosetta has been continuously refactored and extended. Its advantages are its performance and interoperability between broad modeling capabilities. Here we review tools developed in the last 5 years, including over 80 methods. We discuss improvements to the score function, user interfaces and usability. Rosetta is available at http://www.rosettacommons.org.
Assuntos
Substâncias Macromoleculares/química , Modelos Moleculares , Proteínas/química , Software , Simulação de Acoplamento Molecular , Peptidomiméticos/química , Conformação ProteicaRESUMO
Subcellular membraneless assemblies are a reinvigorated area of study in biology, with spirited scientific discussions on the forces between the low-complexity protein domains within these assemblies. To illuminate these forces, we determined the atomic structures of five segments from protein low-complexity domains associated with membraneless assemblies. Their common structural feature is the stacking of segments into kinked ß sheets that pair into protofilaments. Unlike steric zippers of amyloid fibrils, the kinked sheets interact weakly through polar atoms and aromatic side chains. By computationally threading the human proteome on our kinked structures, we identified hundreds of low-complexity segments potentially capable of forming such interactions. These segments are found in proteins as diverse as RNA binders, nuclear pore proteins, and keratins, which are known to form networks and localize to membraneless assemblies.
Assuntos
Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Humanos , Proteoma/químicaRESUMO
The atomic structure of the infectious, protease-resistant, ß-sheet-rich and fibrillar mammalian prion remains unknown. Through the cryo-EM method MicroED, we reveal the sub-ångström-resolution structure of a protofibril formed by a wild-type segment from the ß2-α2 loop of the bank vole prion protein. The structure of this protofibril reveals a stabilizing network of hydrogen bonds that link polar zippers within a sheet, producing motifs we have named 'polar clasps'.
Assuntos
Amiloide/química , Microscopia Crioeletrônica , Ligação de Hidrogênio , Príons/química , Proteínas Amiloidogênicas/química , Animais , Carbamazepina/química , Bovinos , Cricetinae , Cervos , Elétrons , Humanos , Camundongos , Peptídeos/química , Filogenia , Estrutura Secundária de Proteína , Proteoma , Ovinos , Propriedades de Superfície , Difração de Raios XRESUMO
Fibrils and oligomers are the aggregated protein agents of neuronal dysfunction in ALS diseases. Whereas we now know much about fibril architecture, atomic structures of disease-related oligomers have eluded determination. Here, we determine the corkscrew-like structure of a cytotoxic segment of superoxide dismutase 1 (SOD1) in its oligomeric state. Mutations that prevent formation of this structure eliminate cytotoxicity of the segment in isolation as well as cytotoxicity of the ALS-linked mutants of SOD1 in primary motor neurons and in a Danio rerio (zebrafish) model of ALS. Cytotoxicity assays suggest that toxicity is a property of soluble oligomers, and not large insoluble aggregates. Our work adds to evidence that the toxic oligomeric entities in protein aggregation diseases contain antiparallel, out-of-register ß-sheet structures and identifies a target for structure-based therapeutics in ALS.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Superóxido Dismutase-1/metabolismo , Esclerose Lateral Amiotrófica/genética , Animais , Cristalografia por Raios X/métodos , Camundongos , Neurônios Motores/metabolismo , Mutação/genética , Conformação Proteica em Folha beta , Superóxido Dismutase-1/genéticaRESUMO
It has long been known that toxins produced by Bacillus thuringiensis (Bt) are stored in the bacterial cells in crystalline form. Here we describe the structure determination of the Cry3A toxin found naturally crystallized within Bt cells. When whole Bt cells were streamed into an X-ray free-electron laser beam we found that scattering from other cell components did not obscure diffraction from the crystals. The resolution limits of the best diffraction images collected from cells were the same as from isolated crystals. The integrity of the cells at the moment of diffraction is unclear; however, given the short time (â¼ 5 µs) between exiting the injector to intersecting with the X-ray beam, our result is a 2.9-Å-resolution structure of a crystalline protein as it exists in a living cell. The study suggests that authentic in vivo diffraction studies can produce atomic-level structural information.
Assuntos
Bacillus thuringiensis/química , Proteínas de Bactérias/química , Cristalografia por Raios X/métodos , Endotoxinas/química , Proteínas Hemolisinas/química , Esporos Bacterianos/química , Bacillus thuringiensis/ultraestrutura , Toxinas de Bacillus thuringiensis , Cristalização , Cristalografia por Raios X/instrumentação , Lasers , Esporos Bacterianos/ultraestrutura , Síncrotrons , Difração de Raios XRESUMO
The success of macromolecular crystallization depends on the protein's ability to form specific, cohesive intermolecular interactions that serve as crystal contacts. In the cases where the protein lacks surface patches conducive to such interactions, crystallization may not occur. However, it is possible to enhance the likelihood of crystallization by engineering such patches through site-directed mutagenesis, targeting specifically residues with high side chain entropy and replacing them with small amino acids (i.e., surface entropy reduction, SER). This method has proven successful in hundreds of crystallographic analyses of proteins otherwise recalcitrant to crystallization. Three representative cases of the application of the SER strategy, assisted by the automated prediction of the mutation sites using the SER prediction (SERp) server are described.
Assuntos
Biologia Molecular/métodos , Conformação Proteica , Proteínas/química , Aminoácidos/química , Cristalização , Cristalografia por Raios X , Entropia , Mutagênese Sítio-Dirigida , Mutação , Proteínas/genética , Propriedades de SuperfícieRESUMO
Docking Approach using Ray Casting (DARC) is structure-based computational method for carrying out virtual screening by docking small-molecules into protein surface pockets. In a complementary study we find that DARC can be used to identify known inhibitors from large sets of decoy compounds, and can identify new compounds that are active in biochemical assays. Here, we describe our adaptation of DARC for use on Graphics Processing Units (GPUs), leading to a speedup of approximately 27-fold in typical-use cases over the corresponding calculations carried out using a CPU alone. This dramatic speedup of DARC will enable screening larger compound libraries, screening with more conformations of each compound, and including multiple receptor conformations when screening. We anticipate that all three of these enhanced approaches, which now become tractable, will lead to improved screening results.
Assuntos
Gráficos por Computador , Simulação de Acoplamento Molecular , Proteínas/química , Bibliotecas de Moléculas Pequenas/química , Software , Algoritmos , Ensaios de Triagem em Larga Escala , Ligantes , Ligação Proteica , Conformação ProteicaRESUMO
Genomic analyses have identified segments with high fiber-forming propensity in many proteins not known to form amyloid. Proteins are often protected from entering the amyloid state by molecular chaperones that permit them to fold in isolation from identical molecules; but, how do proteins self-chaperone their folding in the absence of chaperones? Here, we explore this question with the stable protein ribonuclease A (RNase A). We previously identified fiber-forming segments of amyloid-related proteins and demonstrated that insertion of these segments into the C-terminal hinge loop of nonfiber-forming RNase A can convert RNase A into the amyloid state through three-dimensional domain-swapping, where the inserted fiber-forming segments interact to create a steric zipper spine. In this study, we convert RNase A into amyloid-like fibers by increasing the loop length and hence conformational freedom of an endogenous fiber-forming segment, SSTSAASS, in the N-terminal hinge loop. This is accomplished by sandwiching SSTSAASS between inserted Gly residues. With these inserts, SSTSAASS is now able to form the steric zipper spine, allowing RNase A to form amyloid-like fibers. We show that these fibers contain RNase A molecules retaining their enzymatic activity and therefore native-like structure. Thus, RNase A appears to prevent fiber formation by limiting the conformational freedom of this fiber-forming segment from entering a steric zipper. Our observations suggest that proteins have evolved to self-chaperone by using similar protective mechanisms.
Assuntos
Amiloide/química , Amiloide/metabolismo , Ribonuclease Pancreático/química , Ribonuclease Pancreático/metabolismo , Animais , Bovinos , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
Amyloid-beta (Aß) aggregates are the main constituent of senile plaques, the histological hallmark of Alzheimer's disease. Aß molecules form ß-sheet containing structures that assemble into a variety of polymorphic oligomers, protofibers, and fibers that exhibit a range of lifetimes and cellular toxicities. This polymorphic nature of Aß has frustrated its biophysical characterization, its structural determination, and our understanding of its pathological mechanism. To elucidate Aß polymorphism in atomic detail, we determined eight new microcrystal structures of fiber-forming segments of Aß. These structures, all of short, self-complementing pairs of ß-sheets termed steric zippers, reveal a variety of modes of self-association of Aß. Combining these atomic structures with previous NMR studies allows us to propose several fiber models, offering molecular models for some of the repertoire of polydisperse structures accessible to Aß. These structures and molecular models contribute fundamental information for understanding Aß polymorphic nature and pathogenesis.
Assuntos
Peptídeos beta-Amiloides/química , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/ultraestrutura , Cristalização , Cristalografia por Raios X , Humanos , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/ultraestrutura , Multimerização Proteica , Estrutura Secundária de ProteínaRESUMO
A very small number of natural proteins have folded configurations in which the polypeptide backbone is knotted. Relatively little is known about the folding energy landscapes of such proteins, or how they have evolved. We explore those questions here by designing a unique knotted protein structure. Biophysical characterization and X-ray crystal structure determination show that the designed protein folds to the intended configuration, tying itself in a knot in the process, and that it folds reversibly. The protein folds to its native, knotted configuration approximately 20 times more slowly than a control protein, which was designed to have a similar tertiary structure but to be unknotted. Preliminary kinetic experiments suggest a complicated folding mechanism, providing opportunities for further characterization. The findings illustrate a situation where a protein is able to successfully traverse a complex folding energy landscape, though the amino acid sequence of the protein has not been subjected to evolutionary pressure for that ability. The success of the design strategy--connecting two monomers of an intertwined homodimer into a single protein chain--supports a model for evolution of knotted structures via gene duplication.
Assuntos
Conformação Proteica , Dobramento de Proteína , Proteínas/química , Proteínas/metabolismo , Biologia Computacional , Cristalografia por Raios X , Cinética , Desnaturação Proteica , Redobramento de Proteína , Estrutura Terciária de Proteína , TermodinâmicaRESUMO
The amylome is the universe of proteins that are capable of forming amyloid-like fibrils. Here we investigate the factors that enable a protein to belong to the amylome. A major factor is the presence in the protein of a segment that can form a tightly complementary interface with an identical segment, which permits the formation of a steric zipper-two self-complementary beta sheets that form the spine of an amyloid fibril. Another factor is sufficient conformational freedom of the self-complementary segment to interact with other molecules. Using RNase A as a model system, we validate our fibrillogenic predictions by the 3D profile method based on the crystal structure of NNQQNY and demonstrate that a specific residue order is required for fiber formation. Our genome-wide analysis revealed that self-complementary segments are found in almost all proteins, yet not all proteins form amyloids. The implication is that chaperoning effects have evolved to constrain self-complementary segments from interaction with each other.
Assuntos
Amiloide/química , Análise de Sequência de Proteína/métodos , Sequência de Aminoácidos , Amiloide/genética , Cristalografia , Estudo de Associação Genômica Ampla , Chaperonas Moleculares/química , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Secundária de Proteína , Ribonuclease Pancreático/química , Ribonuclease Pancreático/genéticaRESUMO
In prion inheritance and transmission, strains are phenotypic variants encoded by protein 'conformations'. However, it is unclear how a protein conformation can be stable enough to endure transmission between cells or organisms. Here we describe new polymorphic crystal structures of segments of prion and other amyloid proteins, which offer two structural mechanisms for the encoding of prion strains. In packing polymorphism, prion strains are encoded by alternative packing arrangements (polymorphs) of beta-sheets formed by the same segment of a protein; in segmental polymorphism, prion strains are encoded by distinct beta-sheets built from different segments of a protein. Both forms of polymorphism can produce enduring conformations capable of encoding strains. These molecular mechanisms for transfer of protein-encoded information into prion strains share features with the familiar mechanism for transfer of nucleic acid-encoded information into microbial strains, including sequence specificity and recognition by noncovalent bonds.
Assuntos
Amiloide/química , Príons/química , Sequência de Aminoácidos , Amiloide/ultraestrutura , Animais , Cristalografia por Raios X , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Camundongos , Microscopia Eletrônica , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de ProteínaRESUMO
beta-Cell dysfunction is an important factor in the development of hyperglycemia of type-2 diabetes mellitus, and pancreatic islet amyloidosis (IA) has been postulated to be one of the main contributors to impaired insulin secretion. The aim of this study was to evaluate the correlation of IA with metabolic parameters and its effect on islets of Langerhans remodeling and relative endocrine-cell volume in baboons. We sequenced the amylin peptide, determined the fibrillogenic propensities, and evaluated pancreatic histology, clinical and biochemical characteristics, and endocrine cell proliferation and apoptosis in 150 baboons with different metabolic status. Amylin sequence in the baboon was 92% similar to humans and showed superimposable fibrillogenic propensities. IA severity correlated with fasting plasma glucose (FPG) (r = 0.662, P < 0.001) and HbA1c (r = 0.726, P < 0.001), as well as with free fatty acid, glucagon values, decreased homeostasis model assessment (HOMA) insulin resistance, and HOMA-B. IA severity was associated with a decreased relative beta-cell volume, and increased relative alpha-cell volume and hyperglucagonemia. These results strongly support the concept that IA and beta-cell apoptosis in concert with alpha-cell proliferation and hypertrophy are key determinants of islets of Langerhans "dysfunctional remodeling" and hyperglycemia in the baboon, a nonhuman primate model of type-2 diabetes mellitus. The most important determinants of IA were age and FPG (R(2) = 0.519, P < 0.0001), and different FPG levels were sensitive and specific to predict IA severity. Finally, a predictive model for islet amyloid severity was generated with age and FPG as required variables.
Assuntos
Amiloidose/patologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/patologia , Amiloide/metabolismo , Animais , Apoptose , Glicemia/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/patologia , Ácidos Graxos/metabolismo , Feminino , Resistência à Insulina , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Masculino , PapioRESUMO
Growing well-diffracting crystals constitutes a serious bottleneck in structural biology. A recently proposed crystallization methodology for "stubborn crystallizers" is to engineer surface sequence variants designed to form intermolecular contacts that could support a crystal lattice. This approach relies on the concept of surface entropy reduction (SER), i.e., the replacement of clusters of flexible, solvent-exposed residues with residues with lower conformational entropy. This strategy minimizes the loss of conformational entropy upon crystallization and renders crystallization thermodynamically favorable. The method has been successfully used to crystallize more than 15 novel proteins, all stubborn crystallizers. But the choice of suitable sites for mutagenesis is not trivial. Herein, we announce a Web server, the surface entropy reduction prediction server (SERp server), designed to identify mutations that may facilitate crystallization. Suggested mutations are predicted based on an algorithm incorporating a conformational entropy profile, a secondary structure prediction, and sequence conservation. Minor considerations include the nature of flanking residues and gaps between mutation candidates. While designed to be used with default values, the server has many user-controlled parameters allowing for considerable flexibility. Within, we discuss (1) the methodology of the server, (2) how to interpret the results, and (3) factors that must be considered when selecting mutations. We also attempt to benchmark the server by comparing the server's predictions with successful SER structures. In most cases, the structure yielding mutations were easily identified by the SERp server. The server can be accessed at http://www.doe-mbi.ucla.edu/Services/SER.
Assuntos
Cristalografia por Raios X , Mutação , Proteínas/química , Proteínas/genética , Software , Sequência de Aminoácidos , Animais , Sequência Conservada , Cristalização , Entropia , Humanos , Internet , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Propriedades de Superfície , Interface Usuário-ComputadorRESUMO
Amyloid diseases, including Alzheimer's and prion diseases, are each associated with unbranched protein fibrils. Each fibril is made of a particular protein, yet they share common properties. One such property is nucleation-dependent fibril growth. Monomers of amyloid-forming proteins can remain in dissolved form for long periods, before rapidly assembly into fibrils. The lag before growth has been attributed to slow kinetics of formation of a nucleus, on which other molecules can deposit to form the fibril. We have explored the energetics of fibril formation, based on the known molecular structure of a fibril-forming peptide from the yeast prion, Sup35, using both classical and quantum (density functional theory) methods. We find that the energetics of fibril formation for the first three layers are cooperative using both methods. This cooperativity is consistent with the observation that formation of amyloid fibrils involves slow nucleation and faster growth.