RESUMO
Pin1 is a peptidyl-prolyl cis-trans isomerase, whose WW domain specifically recognizes the pSer/Thr-Pro motif. Pin1 is involved in multiple phosphorylation events that regulate the activities of various substrates, and Pin1 deregulation has been reported in various diseases, including cancer and Alzheimer's disease. The WW domain of Pin1 has been used as a small model protein to investigate the folding mechanisms of the ß-sheet structure by studying the effect of mutations or its naturally occurring variants. However, only a few studies have investigated the structure and binding of Pin1 WW mutants. In the present work, two naturally occurring Pin1 WW variants, namely, G20D and S16R, derived from the cynomolgus monkey and African green monkey, respectively, were selected to investigate the influence of charge mutation on the structure, stability, and binding properties of the Pin1 WW domain. Analysis using a combination of nuclear magnetic resonance (NMR) and chemical shift-based calculations revealed that the G20D and S16R mutants had high structural similarity to the wild-type Pin1 WW domain. However, the presence of a charge mutation significantly decreased the stability of the Pin1 WW domain. Both the wild-type and G20D forms of the Pin1 WW domain utilized a three-site mode to bind to a phosphorylated Tau peptide, pT231, whereas the S16R mutant binds to the pT231 peptide either in a non-specific manner or through a totally different binding mechanism. Correspondingly, the wild-type and two mutant Pin1 WW domains showed different binding affinities to the Tau phosphopeptide. Considering that the WW domain participates in the catalytic activity of the Pin1 isomerase, our study represents a novel approach for studying Pin1 function through the analysis of its naturally occurring mutants.
Assuntos
Modelos Químicos , Modelos Moleculares , Peptidilprolil Isomerase de Interação com NIMA/química , Peptidilprolil Isomerase de Interação com NIMA/genética , Sítios de Ligação , Simulação por Computador , Ativação Enzimática , Estabilidade Enzimática , Mutação , Peptidilprolil Isomerase de Interação com NIMA/ultraestrutura , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Eletricidade Estática , Relação Estrutura-AtividadeRESUMO
The structures of DNA G-quadruplexes are essential for their functions in vivo and in vitro. Our present study revealed that sequential order of the three G-quadruplex loops, that is, loop transposition, could be a critical factor to determinate the G-quadruplex conformation and consequently improved the catalytic function of G-quadruplex based DNAzyme. In the presence of 100mM K+, loop transposition induced one of the G-quadruplex isomers which shared identical loops but differed in the sequential order of loops into a hybrid topology while the others into predominately parallel topologies. 1D NMR spectroscopy and mutation analysis suggested that the hydrogen bonding from loops residues with nucleotides in flanking sequences may be responsible for the stabilization of the different conformations. A well-known DNAzyme consisting of G-quadruplex and hemin (Ferriprotoporphyrin IX chloride) was chosen to test the catalytic function. We found that the loop transposition could enhance the reaction rate obviously by increasing the hemin binding affinity to G-quadruplex. These findings disclose the relations between the loop transposition, G-quadruplex conformation and catalytic function of DNAzyme.
Assuntos
Biocatálise , DNA Catalítico/fisiologia , Quadruplex G , Hemina/metabolismo , Ligação de HidrogênioRESUMO
Two-dimensional NMR spectroscopies are one of the most frequently used techniques for the structural determination of carbohydrates. However, the data analysis is challenging because of the signal overlap in the 1H homonuclear correlation spectra. We attempted to explore a general strategy for the structural determination of carbohydrates by combined multi-dimensional spectroscopies. The strategy was applied to a human milk oligosaccharide lacto-N-difucohexaose I, that has been previously studied by conventional two-dimensional NMR spectroscopy. Assignment of the intra-residue resonances of the hexasaccharide using the three-dimensional spectrum was straightforward. Consequently, data analysis of the multi-dimensional spectra was significantly simplified, leading to a quicker determination of the intra- and inter-residue connections in the hexasaccharide. Application of the NMR strategy to chondroitin sulfate from bovine cartilage revealed two repeating disaccharide regions of the A and C units of chondroitin sulfate, indicating the high potential of this technique for the structural determination of complex polysaccharides.
Assuntos
Sulfatos de Condroitina/análise , Oligossacarídeos/análise , Animais , Sequência de Carboidratos , Cartilagem/química , Bovinos , Sulfatos de Condroitina/química , Humanos , Leite Humano/química , Ressonância Magnética Nuclear Biomolecular/métodos , Oligossacarídeos/químicaRESUMO
One of the neuropathological features of Alzheimer's disease (AD) is the misfolding of amyloid-ß to form amyloid aggregates, a process highly associated with biological membranes. However, how molecular chirality affects the amyloid formation on phospholipid surfaces has seldom been reported. Here, l- and d-aspartic acid-modified 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (l-/d-Asp-DPPE) is synthesized to construct chiral phospholipid bilayers. We discover that the l-Asp-DPPE liposomes slightly inhibit the Aß(1-40) nucleation process but cannot affect the oligomer elongation process. By contrast, the d-Asp-DPPE liposomes strongly inhibit both nucleation and elongation of the peptide. Notably, l- and d-Asp-DPPE liposomes not only have good biocompatibility but can also rescue Aß(1-40)-aggregation induced cytotoxicity with significant chiral discrimination, in which the cell viability is higher in the presence of d-Asp-DPPE liposomes. Mechanism analysis and molecular dynamics simulation clearly demonstrate that differential electrostatic interactions of Lys16 in Aß(1-40) with l- or d-Asp on the phospholipid contribute to the remarkable chiral discrimination. This study provides a deeper understanding of the crucial amyloidosis process from the perspective of the chiral interface and reveals that the convergence of d-amino acids with the liposomes might be a feasible route for AD prevention.
RESUMO
Atu4866 is a 79-residue conserved hypothetical protein of unknown function from Agrobacterium tumefaciens. Protein sequence alignments show that it shares > or =60% sequence identity with 20 other hypothetical proteins of bacterial origin. However, the structures and functions of these proteins remain unknown so far. To gain insight into the function of this family of proteins, we have determined the structure of Atu4866 as a target of a structural genomics project using solution NMR spectroscopy. Our results reveal that Atu4866 adopts a streptavidin-like fold featuring a beta-barrel/sandwich formed by eight antiparallel beta-strands. Further structural analysis identified a continuous patch of conserved residues on the surface of Atu4866 that may constitute a potential ligand-binding site.
Assuntos
Agrobacterium tumefaciens/química , Proteínas de Bactérias/química , Estreptavidina/química , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Dobramento de ProteínaRESUMO
Thyroid cancer 1 (TC-1) is a 106-residue naturally disordered protein that has been found to associate with thyroid, gastric, and breast cancers. Recent studies showed that the protein functions as a positive regulator in the Wnt/beta-catenin signaling pathway, a pathway that is known to play essential roles in developmental processes and causes tumor formation when misregulated. By competing with beta-catenin for binding to Chibby (Cby), a conserved nuclear protein that antagonizes the beta-catenin-mediated transcriptions, TC-1 up-regulates a number of beta-catenin target genes that are known to be involved in the aggressive behavior of cancers. In order to gain a molecular understanding of the role TC-1 plays in regulating the Wnt/beta-catenin signaling pathway, detailed structural studies of the protein and its interaction with Cby are essential. In this work, we used nuclear magnetic resonance (NMR) spectroscopy to elucidate the structure of TC-1 and its interaction with Cby. Our results indicate that even though TC-1 is naturally disordered, the protein adopts fairly compact conformations under nondenaturing conditions. Chemical shift analysis and relaxation measurements show that three regions (D44-R53, K58-A64, and D73-T88) with high-helical propensity are present in the C-terminal portion of TC-1. Upon addition of Cby, significant broadening of resonance signals derived from these helical regions of TC-1 was observed. The result indicates that the intrinsically disordered TC-1 interacts with Cby via its transient helical structure.
Assuntos
Proteínas de Transporte/química , Regulação Neoplásica da Expressão Gênica , Espectroscopia de Ressonância Magnética/métodos , Proteínas de Neoplasias/química , Proteínas Nucleares/química , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Transdução de Sinais , Temperatura , beta Catenina/metabolismoRESUMO
The targeting of type III secretion (TTS) proteins at the injectisome is an important process in bacterial virulence. Nevertheless, how the injectisome specifically recognizes TTS substrates among all bacterial proteins is unknown. A TTS peripheral membrane ATPase protein located at the base of the injectisome has been implicated in the targeting process. We have investigated the targeting of the EspA filament protein and its cognate chaperone, CesAB, to the EscN ATPase of the enteropathogenic E. coli (EPEC). We show that EscN selectively engages the EspA-loaded CesAB but not the unliganded CesAB. Structure analysis revealed that the targeting signal is encoded in a disorder-order structural transition in CesAB that is elicited only upon the binding of its physiological substrate, EspA. Abrogation of the interaction between the CesAB-EspA complex and EscN resulted in severe secretion and infection defects. Additionally, we show that the targeting and secretion signals are distinct and that the two processes are likely regulated by different mechanisms.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Chaperonas Moleculares/química , Sinais Direcionadores de Proteínas , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Transporte ProteicoRESUMO
AF2241 is a hypothetical protein from Archaeoglobus fulgidus and it belongs to the PFam domain of unknown function 369 (DUF369). NMR structural determination reveals that AF2241 adopts a cyclophilin-like fold, with a beta-barrel core composed of eight beta-strands, one alpha-helix, and one 3(10) helix located at each end of the barrel. The protein displays a high structural similarity to TM1367, another member of DUF369 whose structure has been determined recently by X-ray crystallography. Structural similarity search shows that AF2241 also has a high similarity to human cyclophilin A, however, sequence alignment and electrostatic potential analysis reveal that the residues in the PPIase catalytic site of human cyclophilin A are not conserved in AF2241 or TM1367. Instead, a putative active site of AF2241 maps to a negatively charged pocket composed of 9 conserved residues. Our results suggest that although AF2241 adopts the same fold as the human cyclophilin A, it may have distinct biological function.
Assuntos
Proteínas Arqueais/química , Archaeoglobus fulgidus/metabolismo , Ciclofilina A/química , Ressonância Magnética Nuclear Biomolecular/métodos , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Proteômica/métodos , Homologia de Sequência de Aminoácidos , Eletricidade EstáticaRESUMO
The effects of macromolecular crowding on protein stability and folding kinetics have been studied using the recently developed 15N spin relaxation dispersion technique. By applying this method to a redesigned apocytochrome b562, the kinetics and thermodynamics of the protein folding processes in both the presence and the absence of crowding agents have been characterized. The result indicates that, even under the mild crowded environments (in the presence of 85 mg/mL of PEG 20K), the folding rate of the protein can speed up significantly while the unfolded rate remains unchanged within experimental error.
Assuntos
Grupo dos Citocromos b/química , Dobramento de Proteína , Cinética , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular/métodos , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
Solid-state 1H, 29Si NMR and 23Na MQMAS NMR spectroscopy combined with X-ray powder diffraction (XRD) technique was used to study the reaction between layered sodium disilicate SKS-6 and calcium chloride solution with different concentrations. Based on the experimental results the calcium ion extracting mechanism of SKS-6 was demonstrated to be via ion replacement, during which calcium ions replace sodium ions and combine with the non-bridging oxygen in SKS-6 for charge compensation, and simultaneously sodium ions leave the interlayer. This ion-replacement process was greatly influenced by the substitution of sodium ions by protons as well as the calcium ions concentration. Increasing calcium ions concentration favors the replacement of sodium ions by calcium ions, but prevents sodium ions from being substituted by protons.
RESUMO
Three different kinds of silanols, which include isolated silanol, silanol I (with the hydroxyl proton bonded to an oxygen atom in the adjacent layer) and silanol II (with the hydroxyl proton bonded to the non-bridging oxygen at the same silicon atom), are generated during the hydration process of SKS-6 (delta-Na2Si2O5). 1H-1H nuclear Overhauser enhancement spectroscopy reveals that the proton of silanol I has an effective chemical exchange or spin diffusion with the proton of hydrogen-bonded water, while the proton of silanol II is likely far away from the other proton-containing species. 29Si magic angle spinning, 1H-->29Si CP/MAS NMR and 1H-29Si phase-modulated Lee-Goldburg decoupled correlation experiments demonstrate that the local environments of the silicon sites in the final hydrated sample are mainly composed of Q2 [(SiO)2Si(OH)O(-)Na+], Q3 [(SiO)3Si(OH) and (SiO)3SiO(-)Na+] and Q4 [Si(OSi)4] groups.