RESUMO
A protein superfold is a type of protein fold that is observed in at least three distinct, non-homologous protein families. Structural classification studies have revealed a limited number of prevalent superfolds alongside several infrequent occurring folds, and in α/ß type superfolds, the C-terminal ß-strand tends to favor the edge of the ß-sheet, while the N-terminal ß-strand is often found in the middle. The reasons behind these observations, whether they are due to evolutionary sampling bias or physical interactions, remain unclear. This article offers a physics-based explanation for these observations, specifically for pure parallel ß-sheet topologies. Our investigation is grounded in several established structural rules that are based on physical interactions. We have identified "frustration-free topologies" which are topologies that can satisfy all the rules simultaneously. In contrast, topologies that cannot are termed "frustrated topologies." Our findings reveal that frustration-free topologies represent only a fraction of all theoretically possible patterns, these topologies strongly favor positioning the C-terminal ß-strand at the edge of the ß-sheet and the N-terminal ß-strand in the middle, and there is significant overlap between frustration-free topologies and superfolds. We also used a lattice protein model to thoroughly investigate sequence-structure relationships. Our results show that frustration-free structures are highly designable, while frustrated structures are poorly designable. These findings suggest that superfolds are highly designable due to their lack of frustration, and the preference for positioning C-terminal ß-strands at the edge of the ß-sheet is a direct result of frustration-free topologies. These insights not only enhance our understanding of sequence-structure relationships but also have significant implications for de novo protein design.
Assuntos
Modelos Moleculares , Dobramento de Proteína , Proteínas , Proteínas/química , Conformação Proteica em Folha beta , Biologia Computacional/métodosRESUMO
Beta turns, in which the protein backbone abruptly changes direction over four amino acid residues, are the most common type of protein secondary structure after alpha helices and beta sheets and play key structural and functional roles. Previous work has produced classification systems for turn geometry at multiple levels of precision, but these operate in backbone dihedral-angle (Ramachandran) space, and the absence of a local Euclidean-space coordinate system and structural alignment for turns, or of any systematic Euclidean-space characterization of turn backbone shape, presents challenges for the visualization, comparison and analysis of the wide range of turn conformations and the design of turns and the structures that incorporate them. This work derives a turn-local coordinate system that implicitly aligns turns, together with a set of geometric descriptors that characterize the bulk BB shapes of turns and describe modes of structural variation not explicitly captured by existing systems. These modes are shown to be meaningful by the demonstration of clear relationships between descriptor values and the electrostatic energy of the beta-turn H-bond, the overrepresentations of key side-chain motifs, and the structural contexts of turns. Geometric turn descriptors complement Ramachandran-space classifications, and they can be used to select turn structures for compatibility with particular side-chain interactions or contexts. Potential applications include protein design and other tasks in which an enhanced Euclidean-space characterization of turns may improve understanding or performance. The web-based tools ExploreTurns, MapTurns, and ProfileTurn, available at www.betaturn.com, incorporate turn-local coordinates and turn descriptors and demonstrate their utility.
Assuntos
Modelos Moleculares , Proteínas , Proteínas/química , Ligação de Hidrogênio , Bases de Dados de Proteínas , Estrutura Secundária de Proteína , Eletricidade Estática , Conformação Proteica em Folha betaRESUMO
Dynamic attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy at both solutions and coatings of a semicrystalline silk material derived from Bombyx mori was applied to monitor the ß-sheet conformation, which is known to correlate with silk protein crystallinity. The secondary structure-sensitive Amide I band was analyzed. Two silk protein samples were studied: native-based silk buffer fibroin (NSF) was extracted from silk glands and regenerated silk fibroin (RSF) was extracted from degummed cocoons. Solutions of both NSF and RSF at 2 mg/mL featured low initial ß-sheet contents of 5-12%, which further increased to 47-53% after 24 h. RSF and NSF solutions at 23 mg/mL also featured low initial ß-sheet contents of 9-10%, which yet only slightly increased to 16-17% after 24 h. Coatings deposited from RSF solutions showed high surface integrity (Q > 99%) after rinsing in mineralized water, enabling interfacial drug delivery applications. RSF coatings were post-treated with either formic acid (FA) or pure methanol (MeOH) vapor to showcase inducibility of crystalline domains in RSF coatings. Such coatings were loaded with the model antibiotic drugs tetracycline (TCL) and streptomycin (STRP), and the sustained release of TCL was followed in contact with (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) buffer. RSF/TCL coatings post-treated with formic acid (FA) vapor followed by methanol (MeOH) vapor showed a significantly lower (52%) initial burst of rather hydrophobic TCL compared to untreated RSF/TCL coatings (72%), while no such significant release difference was observed for hydrophilic STRP. This was rationalized by a specific interaction between nonpolar TCL and hydrophobic crystalline RSF domains.
Assuntos
Bombyx , Fibroínas , Animais , Bombyx/química , Espectroscopia de Infravermelho com Transformada de Fourier , Fibroínas/química , Conformação Proteica em Folha beta , Liberação Controlada de Fármacos , Seda/química , Soluções , Materiais Revestidos Biocompatíveis/química , Tetraciclina/químicaRESUMO
Methods from artificial intelligence (AI), in general, and machine learning, in particular, have kept conquering new territories in numerous areas of science. Most of the applications of these techniques are restricted to the classification of large data sets, but new scientific knowledge can seldom be inferred from these tools. Here we show that an AI-based amyloidogenecity predictor can strongly differentiate the border- and the internal hexamers of ß-pleated sheets when screening all the Protein Data Bank-deposited homology-filtered protein structures. Our main result shows that more than 30% of internal hexamers of ß sheets are predicted to be amyloidogenic, while just outside the border regions, only 3% are predicted as such. This result may elucidate a general protection mechanism of proteins against turning into amyloids: if the borders of ß-sheets were amyloidogenic, then the whole ß sheet could turn more easily into an insoluble amyloid-structure, characterized by periodically repeated parallel ß-sheets. We also present that no analogous phenomenon exists on the borders of α-helices or randomly chosen subsequences of the studied protein structures.
Assuntos
Amiloide , Amiloide/química , Humanos , Conformação Proteica em Folha beta , Bases de Dados de Proteínas , Modelos Moleculares , Inteligência Artificial , Estrutura Secundária de ProteínaRESUMO
Klebsiella pneumoniae (Kp) is an infectious disease pathogen that poses a significant global health threat due to its potential to cause severe infections and its tendency to exhibit multidrug resistance. Understanding the enzymatic mechanisms of the oxygen-insensitive nitroreductases (Kp-NRs) from Kp is crucial for the development of effective nitrofuran drugs, such as nitrofurantoin, that can be activated as antibiotics. In this paper, three crystal structures of two Kp-NRs (PDB entries 7tmf/7tmg and 8dor) are presented, and an analysis of their crystal structures and their flavin mononucleotide (FMN)-binding mode is provided. The structures with PDB codes 7tmf (Kp-NR1a), 7tmg (Kp-NR1b) and 8dor (Kp-NR2) were determined at resolutions of 1.97, 1.90 and 1.35â Å, respectively. The Kp-NR1a and Kp-NR1b structures adopt an αß fold, in which four-stranded antiparallel ß-sheets are surrounded by five helices. With domain swapping, the ß-sheet was expanded with a ß-strand from the other molecule of the dimer. The difference between the structures lies in the loop spanning Leu173-Ala185: in Kp-NR1a the loop is disordered, whereas the loop adopts multiple conformations in Kp-NR1b. The FMN interactions within Kp-NR1/NR2 involve hydrogen-bond and π-stacking interactions. Kp-NR2 contains four-stranded antiparallel ß-sheets surrounded by eight helices with two short helices and one ß-sheet. Structural and sequence alignments show that Kp-NR1a/b and Kp-NR2 are homologs of the Escherichia coli oxygen-insensitive NRs YdjA and NfnB and of Enterobacter cloacae NR, respectively. By homology inference from E. coli, Kp-NR1a/b and Kp-NR2 may detoxify polynitroaromatic compounds and Kp-NR2 may activate nitrofuran drugs to cause bactericidal activity through a ping-pong bi-bi mechanism, respectively.
Assuntos
Klebsiella pneumoniae , Modelos Moleculares , Nitrorredutases , Klebsiella pneumoniae/enzimologia , Cristalografia por Raios X , Nitrorredutases/química , Nitrorredutases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Sequência de Aminoácidos , Mononucleotídeo de Flavina/metabolismo , Mononucleotídeo de Flavina/química , Sítios de Ligação , Ligação Proteica , Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/enzimologia , Conformação Proteica em Folha beta , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genéticaRESUMO
The secondary structures of polypeptides, such as an α-helix and a ß-sheet, often impart specific properties and functions, making the regulation of their secondary structures of great significance. Particularly, water-soluble polypeptides bearing a ß-sheet conformation are rare and challenging to achieve. Here, a series of oligo(ethylene glycol)-modified lysine N-carboxylic anhydrides (EGmK-NCA, where m = 1-3) and the corresponding polymers EGmKn are synthesized, with urethane bonds as the linker between the side-chain EG and lysine. The secondary structure of EGmKn is delicately regulated by both m and n, the length (number of repeating units) of EG and the degree of polymerization (DP), respectively. Among them, EG2Kn adopts a ß-sheet conformation with good water solubility at an appropriate DP and forms physically cross-linked hydrogels at a concentration as low as 1 wt %. The secondary structures of EG1Kn can be tuned by DP, exhibiting either a ß-sheet or an α-helix, whereas EG3Kn appears to a adopt pure and stable α-helix with no dependence on DP. Compared to previous works reporting EG-modified lysine-derived polypeptides bearing exclusively an α-helix conformation, this work highlights the important and unexpected role of the urethane connecting unit and provides useful case studies for understanding the secondary structure of polypeptides.
Assuntos
Peptídeos , Conformação Proteica em Folha beta , Solubilidade , Água , Peptídeos/química , Água/química , Polietilenoglicóis/química , Lisina/química , Hidrogéis/química , Etilenoglicol/química , Estrutura Secundária de Proteína , PolimerizaçãoRESUMO
In a recent issue of Nature Chemical Biology, Emmanouilidis et al. (2024) investigate the maturation of biomolecular condensates of FUS1-267 and probe the molecular details of droplet aging. They observe that the liquid-to-solid transition of the droplet is mediated at the surface by FUS1-267 molecules that have adopted ß-strand conformations.
Assuntos
Proteína FUS de Ligação a RNA , Proteína FUS de Ligação a RNA/metabolismo , Proteína FUS de Ligação a RNA/química , Biofísica , Condensados Biomoleculares/metabolismo , Condensados Biomoleculares/química , Humanos , Conformação Proteica em Folha betaRESUMO
Peptide self-assembly into amyloid fibrils provides numerous applications in drug delivery and biomedical engineering applications. We augment our previously-established computational screening technique along with experimental biophysical characterization to discover 7-mer peptides that self-assemble into "parallel ß-sheets", that is, ß-sheets with N-terminus-to-C-terminus ð½-strand vectors oriented in parallel. To accomplish the desired ß-strand organization, we applied the PepAD amino acid sequence design software to the Class-1 cross-ß spine defined by Sawaya et al. This molecular configuration includes two layers of parallel ß-sheets stacked such that N-terminus-to-C-terminus vectors are oriented antiparallel for molecules on adjacent ß-sheets. The first cohort of PepAD identified peptides were examined for their fibrillation behavior in DMD/PRIME20 simulations, and the top performing sequence was selected as a prototype for a subsequent round of sequence refinement. The two rounds of design resulted in a library of eight 7-mer peptides. In DMD/PRIME20 simulations, five of these peptides spontaneously formed fibril-like structures with a predominantly parallel ð½-sheet arrangement, two formed fibril-like structure with <50% in parallel ð½-sheet arrangement and one remained a random coil. Among the eight candidate peptides produced by PepAD and DMD/PRIME20, five were synthesized and purified. All five assembled into amyloid fibrils composed of parallel ß-sheets based on Fourier transform infrared spectroscopy, circular dichroism, electron microscopy, and thioflavin-T fluorescence spectroscopy measurements.
Assuntos
Método de Monte Carlo , Conformação Proteica em Folha beta , Nanofibras/química , Peptídeos/química , Sequência de Aminoácidos , Estrutura Secundária de Proteína , Amiloide/química , Modelos Moleculares , Simulação de Dinâmica MolecularRESUMO
Affilin proteins, artificial binding proteins based on the ubiquitin scaffold, have been generated by directed protein evolution to yield de-novo variants that bind the extra-domain B (EDB) of oncofetal fibronectin, an established marker of tumor neovasculature. The crystal structures of two EDB-specific Affilin variants reveal a striking structural plasticity of the ubiquitin scaffold, characterised by ß-strand slippage, leading to different negative register shifts of the ß5 strands. This process recruits amino acid residues from ß5 towards the N-terminus to an adjacent loop region and subsequent residues into ß5, respectively, remodeling the binding interface and leading to target specificity and affinity. Protein backbone alterations resulting from ß-strand register shifts, as seen in the ubiquitin fold, can pose additional challenges to protein engineering as structural evidence of these events is still limited and they are difficult to predict. However, they can surface under the selection pressure of directed evolution and suggest that backbone plasticity allowing ß-strand slippages can increase structural diversity, enhancing the evolutionary potential of a protein scaffold.
Assuntos
Fibronectinas , Ubiquitina , Fibronectinas/metabolismo , Fibronectinas/química , Fibronectinas/genética , Ubiquitina/metabolismo , Humanos , Ligação Proteica , Conformação Proteica em Folha beta , Modelos Moleculares , Cristalografia por Raios X , Engenharia de ProteínasRESUMO
Although hydrophobic interactions provide the main driving force for initial peptide aggregation, their role in regulating suprastructure handedness of higher-order architectures remains largely unknown. We here interrogate the effects of hydrophobic amino acids on handedness at various assembly stages of peptide amphiphiles. Our studies reveal that relative to aliphatic side chains, aromatic side chains set the twisting directions of single ß-strands due to their strong steric repulsion to the backbone, and upon packing into multi-stranded ß-sheets, the side-chain aromatic interactions between strands form the aromatic ladders with a directional preference. This ordering not only leads to parallel ß-sheet arrangements but also induces the chiral flipping over of single ß-strands within a ß-sheet. In contrast, the lack of orientational hydrophobic interactions in the assembly of aliphatic peptides implies no chiral inversion upon packing into ß-sheets. This study opens an avenue to harness peptide aggregates with targeted handedness via aromatic side-chain interactions.
Assuntos
Interações Hidrofóbicas e Hidrofílicas , Peptídeos , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica em Folha beta , Estereoisomerismo , Estrutura Secundária de Proteína , Aminoácidos Aromáticos/química , Dicroísmo Circular , Modelos Moleculares , Aminoácidos/químicaRESUMO
Transmembrane ß-barrels have considerable potential for a broad range of sensing applications. Current engineering approaches for nanopore sensors are limited to naturally occurring channels, which provide suboptimal starting points. By contrast, de novo protein design can in principle create an unlimited number of new nanopores with any desired properties. Here we describe a general approach to designing transmembrane ß-barrel pores with different diameters and pore geometries. Nuclear magnetic resonance and crystallographic characterization show that the designs are stably folded with structures resembling those of the design models. The designs have distinct conductances that correlate with their pore diameter, ranging from 110 picosiemens (~0.5 nanometer pore diameter) to 430 picosiemens (~1.1 nanometer pore diameter). Our approach opens the door to the custom design of transmembrane nanopores for sensing and sequencing applications.
Assuntos
Nanoporos , Engenharia de Proteínas , Dobramento de Proteína , Cristalografia por Raios X , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica em Folha beta , Modelos MolecularesRESUMO
The effect of ß-sheet ratio and chain length on all-ß proteins was investigated by MD simulations. Protein samples composed of different repeating units with various ß-sheet ratios or a different number of repeating units were simulated under a broad temperature range. The simulation results show that the smaller radius of gyration was achieved by the protein with the higher proportion of ß-sheet secondary structure, which had the lower nonbonded energy with more HBs within the protein. The root mean square deviation (RMSD) and the root mean square fluctuation (RMSF) both increased with temperature, especially in the case of a longer chain. The visible period was also shown according to the repeated secondary structure. Several minimum values of RMSF were located on the skeleton of Cα atoms participating in the ß-sheet, indicating that it is a kind of stable secondary structure. We also concluded that proteins with a short chain or a lower ratio of ß-sheet could easily transform their oriented and compact structures to other ones, such as random coils, turns, and even α-helices. These results clarified the relationship from the primary level to the 3D structure of proteins and potentially predicted protein folding.
Assuntos
Simulação de Dinâmica Molecular , Conformação Proteica em Folha beta , Proteínas , Proteínas/química , Dobramento de Proteína , Estrutura Secundária de Proteína , TemperaturaRESUMO
Peptide self-assemblies could leverage their specificity, stability, biocompatibility, and electrochemical activity to create functionalized interfaces for molecular sensing and detection. However, the dynamics within these interfaces are complex, with competing forces, including those maintaining peptide structures, recognizing analytes, and facilitating signal transmission. Such competition could lead to nonspecific interference, compromising the detection sensitivity and accuracy. In this study, a series of peptides with precise structures and controllable electron transfer capabilities were designed. Through examining their stacking patterns, the interplay between the peptides' hierarchical structures, their ability to recognize targets, and their conductivity were clarified. Among these, the EP5 peptide assembly was identified for its ability to form controllable electronic tunnels facilitated by π-stacking induced ß-sheets. EP5 could enhance the long-range conductivity, minimize nonspecific interference, and exhibit targeted recognition capabilities. Based on EP5, an electrochemical sensing interface toward the disease marker PD-L1 (programmed cell death ligand 1) was developed, suitable for both whole blood assay and in vivo companion diagnosis. It opens a new avenue for crafting electrochemical detection interfaces with specificity, sensitivity, and compatibility.
Assuntos
Técnicas Eletroquímicas , Técnicas Eletroquímicas/métodos , Humanos , Conformação Proteica em Folha beta , Peptídeos/química , Antígeno B7-H1/análise , Antígeno B7-H1/sangue , Elétrons , AnimaisRESUMO
Mutations in the protein superoxide dismutase-1 (SOD1) promote its misfolding and aggregation, ultimately causing familial forms of the debilitating neurodegenerative disease amyotrophic lateral sclerosis (ALS). Currently, over 220 (mostly missense) ALS-causing mutations in the SOD1 protein have been identified, indicating that common structural features are responsible for aggregation and toxicity. Using in silico tools, we predicted amyloidogenic regions in the ALS-associated SOD1-G85R mutant, finding seven regions throughout the structure. Introduction of proline residues into ß-strands II (I18P) or III (I35P) reduced the aggregation propensity and toxicity of SOD1-G85R in cells, significantly more so than proline mutations in other amyloidogenic regions. The I18P and I35P mutations also reduced the capability of SOD1-G85R to template onto previously formed non-proline mutant SOD1 aggregates as measured by fluorescence recovery after photobleaching. Finally, we found that, while the I18P and I35P mutants are less structurally stable than SOD1-G85R, the proline mutants are less aggregation-prone during proteasome inhibition, and less toxic to cells overall. Our research highlights the importance of a previously underappreciated SOD1 amyloidogenic region in ß-strand II (15QGIINF20) to the aggregation and toxicity of SOD1 in ALS mutants, and suggests that ß-strands II and III may be good targets for the development of SOD1-associated ALS therapies.
Assuntos
Esclerose Lateral Amiotrófica , Agregados Proteicos , Superóxido Dismutase-1 , Superóxido Dismutase-1/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/química , Humanos , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Mutação , Conformação Proteica em Folha beta , Modelos Moleculares , Prolina/metabolismo , Amiloide/metabolismo , Amiloide/química , Dobramento de ProteínaRESUMO
The effect of halide substitution in Grubbs-Hoveyda II catalysts (GHII catalysts) embedded in the engineered ß-barrel protein nitrobindin (NB4exp) on metathesis activity in aqueous media was studied. Maleimide tagged dibromido and diiodido derivates of the GHII catalyst were synthesized and covalently conjugated to NB4exp. The biohybrid catalysts were characterized spectroscopically confirming the structural integrity. When the two chloride substituents at ruthenium center were exchanged against bromide and iodide, the diiodo derivative was found to show significantly higher catalytic activity in ring-closing metathesis of α,ω-diolefins, whereas the dibromido derivative was less efficient when compared with the parent dichlorido catalyst. Using the diiodido catalyst, high turnover numbers of up to 75 were observed for ring-closing metathesis (RCM) yielding unsaturated six- and seven-membered N-heterocycles.
Assuntos
Alcenos , Catálise , Alcenos/química , Rutênio/química , Conformação Proteica em Folha betaRESUMO
ATTR amyloidosis is caused by deposition of large, insoluble aggregates (amyloid fibrils) of cross-ß-sheet TTR protein molecules on the intercellular surfaces of tissues. The process of amyloid formation from monomeric TTR protein molecules to amyloid deposits has not been fully characterized and is therefore modeled in this paper. Two models are considered: 1) TTR monomers in the blood spontaneously fold into a ß-sheet conformation, aggregate into short proto-fibrils that then circulate in the blood until they find a complementary tissue where the proto-fibrils accumulate to form the large, insoluble amyloid fibrils found in affected tissues. 2) TTR monomers in the native or ß-sheet conformation circulate in the blood until they find a tissue binding site and deposit in the tissue or tissues forming amyloid deposits in situ. These models only differ on where the selection for ß-sheet complementarity occurs, in the blood where wt-wt, wt-v, and v-v interactions determine selectivity, or on the tissue surface where tissue-wt and tissure-v interactions also determine selectivity. Statistical modeling in both cases thus involves selectivity in fibril aggregation and tissue binding. Because binding of protein molecules into fibrils and binding of fibrils to tissues occurs through multiple weak non-covalent bonds, strong complementarity between ß-sheet molecules and between fibrils and tissues is required to explain the insolubility and tissue selectivity of ATTR amyloidosis. Observation of differing tissue selectivity and thence disease phenotypes from either pure wildtype TTR protein or a mix of wildtype and variant molecules in amyloid fibrils evidences the requirement for fibril-tissue complementarity. Understanding the process that forms fibrils and binds fibrils to tissues may lead to new possibilities for interrupting the process and preventing or curing ATTR amyloidosis.
Assuntos
Amiloide , Pré-Albumina , Pré-Albumina/metabolismo , Pré-Albumina/química , Humanos , Amiloide/metabolismo , Amiloide/química , Neuropatias Amiloides Familiares/metabolismo , Neuropatias Amiloides Familiares/patologia , Amiloidose/metabolismo , Modelos Moleculares , Conformação Proteica em Folha betaRESUMO
In silico validation of de novo designed proteins with deep learning (DL)-based structure prediction algorithms has become mainstream. However, formal evidence of the relationship between a high-quality predicted model and the chance of experimental success is lacking. We used experimentally characterized de novo water-soluble and transmembrane ß-barrel designs to show that AlphaFold2 and ESMFold excel at different tasks. ESMFold can efficiently identify designs generated based on high-quality (designable) backbones. However, only AlphaFold2 can predict which sequences have the best chance of experimentally folding among similar designs. We show that ESMFold can generate high-quality structures from just a few predicted contacts and introduce a new approach based on incremental perturbation of the prediction ("in silico melting"), which can reveal differences in the presence of favorable contacts between designs. This study provides a new insight on DL-based structure prediction models explainability and on how they could be leveraged for the design of increasingly complex proteins; in particular membrane proteins which have historically lacked basic in silico validation tools.
Assuntos
Proteínas de Membrana , Dobramento de Proteína , Solubilidade , Proteínas de Membrana/química , Água/química , Simulação por Computador , Modelos Moleculares , Conformação Proteica em Folha beta , Aprendizado Profundo , AlgoritmosRESUMO
Successive cleavages of amyloid precursor protein C-terminal fragment with 99 residues (APP-C99) by γ-secretase result in amyloid-ß (Aß) peptides of varying lengths. Most cleavages have a step size of three residues. To elucidate the underlying mechanism, we determined the atomic structures of human γ-secretase bound individually to APP-C99, Aß49, Aß46, and Aß43. In all cases, the substrate displays the same structural features: a transmembrane α-helix, a three-residue linker, and a ß-strand that forms a hybrid ß-sheet with presenilin 1 (PS1). Proteolytic cleavage occurs just ahead of the substrate ß-strand. Each cleavage is followed by unwinding and translocation of the substrate α-helix by one turn and the formation of a new ß-strand. This mechanism is consistent with existing biochemical data and may explain the cleavages of other substrates by γ-secretase.
Assuntos
Secretases da Proteína Precursora do Amiloide , Peptídeos beta-Amiloides , Precursor de Proteína beta-Amiloide , Presenilina-1 , Humanos , Peptídeos beta-Amiloides/química , Precursor de Proteína beta-Amiloide/química , Secretases da Proteína Precursora do Amiloide/química , Cristalografia por Raios X , Modelos Moleculares , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/química , Presenilina-1/química , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Proteólise , Especificidade por SubstratoRESUMO
The ligand-induced conformational switch of proteins has great significance in understanding the biophysics and biochemistry of their self-assembly. In this work, we have investigated the ability of plumbagin (PL), a hydroxynaphthoquinone compound found in the root of the medicinal plant Plumbago zeylanica, to modulate aggregation precursor state, aggregation kinetics and generate distinct fibril of human serum albumin (HSA). PL was found to moderately bind (binding constant Ka â¼ 10-4 M-1)) to domain-II of HSA in the stoichiometric ratio of 1:1. We found that PL-HSA complex aggregation was accelerated as compared to that of HSA aggregation and it may be through an independent pathway. We also detected that fibril produced in the presence of PL is wider in diameter, contains a higher amount of ß-sheet (â¼18%) and disordered (â¼46%) structures, and is less stable. We concluded that the acceleration of aggregation reaction and generation of fibril polymorphism was mainly because of the higher extent of unfolding and high content of non-native ß-sheet structure in the aggregation precursor state of PL-HSA complex. This study offers opportunities to explore the ability of ligand binding to modulate aggregation reactions and generate polymorphic protein fibrils.
Assuntos
Amiloide , Naftoquinonas , Agregados Proteicos , Naftoquinonas/química , Naftoquinonas/metabolismo , Humanos , Amiloide/química , Amiloide/metabolismo , Cinética , Conformação Proteica em Folha beta , Ligação Proteica , Albumina Sérica Humana/química , Albumina Sérica Humana/metabolismo , Albumina Sérica Humana/genética , Albumina Sérica/química , Albumina Sérica/metabolismoRESUMO
Phosphate plays a vital role in spider silk spinning and has been utilized in numerous artificial silk spinning attempts to replicate the remarkable mechanical properties of natural silk fiber. Its application in artificial processes has, however, yielded varying outcomes. It is thus necessary to investigate the origins and mechanisms behind these differences. By using recombinant silk protein SC-ADF3 derived from the garden spider Araneus diadematus, here, we describe its conformational changes under various conditions, elucidating the effect of phosphate on SC-ADF3 silk protein properties and interactions. Our results demonstrate that elevated phosphate levels induce the irreversible conformational conversion of SC-ADF3 from random coils to ß-sheet structures, leading to decreased protein solubility over time. Furthermore, exposure of SC-ADF3 to phosphate stiffens already formed structures and reduces the ability to form new interactions. Our findings offer insights into the underlying mechanism through which phosphate-induced ß-sheet structures in ADF3-related silk proteins impede fiber formation in the subsequent phases. From a broader perspective, our studies emphasize the significance of silk protein conformation for functional material formation, highlighting that the formation of ß-sheet structures at the initial stages of protein assembly will affect the outcome of material forming processes.