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1.
Biophys J ; 112(8): 1621-1633, 2017 Apr 25.
Artículo en Inglés | MEDLINE | ID: mdl-28445753

RESUMEN

Pyroglutamate-modified amyloid-ß (pEAß) has been described as a relevant Aß species in Alzheimer's-disease-affected brains, with pEAß (3-42) as a dominant isoform. Aß (1-40) and Aß (1-42) have been well characterized under various solution conditions, including aqueous solutions containing trifluoroethanol (TFE). To characterize structural properties of pEAß (3-42) possibly underlying its drastically increased aggregation propensity compared to Aß (1-42), we started our studies in various TFE-water mixtures and found striking differences between the two Aß species. Soluble pEAß (3-42) has an increased tendency to form ß-sheet-rich structures compared to Aß (1-42), as indicated by circular dichroism spectroscopy data. Kinetic assays monitored by thioflavin-T show drastically accelerated aggregation leading to large fibrils visualized by electron microscopy of pEAß (3-42) in contrast to Aß (1-42). NMR spectroscopy was performed for backbone and side-chain chemical-shift assignments of monomeric pEAß (3-42) in 40% TFE solution. Although the difference between pEAß (3-42) and Aß (1-42) is purely N-terminal, it has a significant impact on the chemical environment of >20% of the total amino acid residues, as revealed by their NMR chemical-shift differences. Freshly dissolved pEAß (3-42) contains two α-helical regions connected by a flexible linker, whereas the N-terminus remains unstructured. We found that these α-helices act as a transient intermediate to ß-sheet and fibril formation of pEAß (3-42).


Asunto(s)
Péptidos beta-Amiloides/química , Amiloide/química , Fragmentos de Péptidos/química , Amiloide/metabolismo , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Animales , Benzotiazoles , Dicroismo Circular , Cinética , Microscopía Electrónica de Transmisión , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Agregación Patológica de Proteínas/metabolismo , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Soluciones , Tiazoles/química , Trifluoroetanol/química , Agua/química
2.
Data Brief ; 8: 605-12, 2016 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-27419198

RESUMEN

The third WW domain (WW3*) of human Nedd4-1 (Neuronal precursor cell expressed developmentally down-regulated gene 4-1) interacts with the poly-proline (PY) motifs of the human epithelial Na+ channel (hENaC) subunits at micromolar affinity. This data supplements the article (Panwalkar et al., 2015) [1]. We describe the NMR experiments used to solve the solution structure of the WW3* domain. We also present NOE network data for defining the rotameric state of side chains of peptide binding residues, and complement this data with χ 1 dihedral angles derived from (3) J couplings and molecular dynamics simulations data.

3.
Biochemistry ; 55(4): 659-74, 2016 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-26685112

RESUMEN

The four WW domains of human Nedd4-1 (neuronal precursor cell expressed developmentally downregulated gene 4-1) interact with the PPxY (PY) motifs of the human epithelial Na(+) channel (hENaC) subunits, with the third WW domain (WW3*) showing the highest affinity. We have shown previously that the α-hENaC PY motif binding interface of WW3* undergoes conformational exchange on the millisecond time scale, indicating that conformational sampling plays a role in peptide recognition. To further understand this role, the structure and dynamics of hNedd4-1 WW3* were investigated. The nuclear Overhauser effect-derived structure of apo-WW3* resembles the domain in complex with the α-hENaC peptide, although particular side chain conformations change upon peptide binding, which was further investigated by molecular dynamics simulations. Model-free analysis of the (15)N nuclear magnetic resonance spin relaxation data showed that the apo and peptide-bound states of WW3* have similar backbone picosecond to nanosecond time scale dynamics. However, apo-WW3* exhibits pronounced chemical exchange on the millisecond time scale that is quenched upon peptide binding. (1)HN and (15)N Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments at various temperatures revealed that apo-WW3* exists in an equilibrium between the natively folded peptide binding-competent state and a random coil-like denatured state. The thermodynamics of the folding equilibrium was determined by fitting a thermal denaturation profile monitored by circular dichroism spectroscopy in combination with the CPMG data, leading to the conclusion that the unfolded state is populated to ∼ 20% at 37 °C. These results show that the binding of the hNedd4-1 WW3* domain to α-hENaC is coupled to the folding equilibrium.


Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Simulación de Dinámica Molecular , Ubiquitina-Proteína Ligasas/química , Secuencias de Aminoácidos , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Humanos , Ubiquitina-Proteína Ligasas Nedd4 , Pliegue de Proteína , Estructura Terciaria de Proteína , Ubiquitina-Proteína Ligasas/metabolismo
4.
Biochemistry ; 54(35): 5469-79, 2015 Sep 08.
Artículo en Inglés | MEDLINE | ID: mdl-26284781

RESUMEN

Autophagy is a fundamental homeostatic process in eukaryotic organisms, fulfilling essential roles in development and adaptation to stress. Among other factors, formation of autophagosomes critically depends on proteins of the Atg8 (autophagy-related protein 8) family, which are reversibly conjugated to membrane lipids. We have applied X-ray crystallography, nuclear magnetic resonance spectroscopy, and molecular dynamics simulations to study the conformational dynamics of Atg8-type proteins, using GATE-16 (Golgi-associated ATPase enhancer of 16 kDa), also known as GABARAPL2, as a model system. This combination of complementary approaches provides new insight into a structural transition centered on the C-terminus, which is crucial for the biological activity of these proteins.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Proteínas Adaptadoras Transductoras de Señales/química , Autofagia , Proteínas de Microfilamentos/biosíntesis , Proteínas de Microfilamentos/química , Autofagia/fisiología , Familia de las Proteínas 8 Relacionadas con la Autofagia , Cristalización , Cristalografía por Rayos X , Humanos , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína
5.
Biomol NMR Assign ; 9(2): 243-6, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25324217

RESUMEN

Hyperpolarization activated and cyclic nucleotide-gated (HCN) ion channels as well as cyclic nucleotide-gated (CNG) ion channels are essential for the regulation of cardiac cells, neuronal excitability, and signaling in sensory cells. Both classes are composed of four subunits. Each subunit comprises a transmembrane region, intracellular N- and C-termini, and a C-terminal cyclic nucleotide-binding domain (CNBD). Binding of cyclic nucleotides to the CNBD promotes opening of both CNG and HCN channels. In case of CNG channels, binding of cyclic nucleotides to the CNBD is sufficient to open the channel. In contrast, HCN channels open upon membrane hyperpolarization and their activity is modulated by binding of cyclic nucleotides shifting the activation potential to more positive values. Although several high-resolution structures of CNBDs from HCN and CNG channels are available, the gating mechanism for murine HCN2 channel, which leads to the opening of the channel pore, is still poorly understood. As part of a structural investigation, here, we report the complete backbone and side chain resonance assignments of the murine HCN2 CNBD with part of the C-linker.


Asunto(s)
Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/química , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Resonancia Magnética Nuclear Biomolecular , Nucleótidos Cíclicos/metabolismo , Animales , Ligandos , Ratones , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Espectroscopía de Protones por Resonancia Magnética
6.
Angew Chem Int Ed Engl ; 53(16): 4227-30, 2014 Apr 14.
Artículo en Inglés | MEDLINE | ID: mdl-24623599

RESUMEN

The misfolding and aggregation of the protein α-synuclein (α-syn), which results in the formation of amyloid fibrils, is involved in the pathogenesis of Parkinson's disease and other synucleinopathies. The emergence of amyloid toxicity is associated with the formation of partially folded aggregation intermediates. Here, we engineered a class of binding proteins termed ß-wrapins (ß-wrap proteins) with affinity for α-synuclein (α-syn). The NMR structure of an α-syn:ß-wrapin complex reveals a ß-hairpin of α-syn comprising the sequence region α-syn(37-54). The ß-wrapin inhibits α-syn aggregation and toxicity at substoichiometric concentrations, demonstrating that it interferes with the nucleation of aggregation.


Asunto(s)
alfa-Sinucleína/química , Secuencia de Aminoácidos , Humanos , Espectroscopía de Resonancia Magnética , Enfermedad de Parkinson/metabolismo , Ingeniería de Proteínas , Pliegue de Proteína , Estructura Secundaria de Proteína
7.
Biochemistry ; 52(26): 4460-73, 2013 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-23746326

RESUMEN

In bacteria and fungi, various light, oxygen, voltage (LOV) sensory systems that lack a fused effector domain but instead contain only short N- and C-terminal extensions flanking the LOV core exist. In the prokaryotic kingdom, this so-called "short" LOV protein family represents the third largest LOV photoreceptor family. This observation prompted us to study their distribution and phylogeny as well as their photochemical and structural properties in more detail. We recently described the slow and fast reverting "short" LOV proteins PpSB1-LOV and PpSB2-LOV from Pseudomonas putida KT2440 whose adduct state lifetimes varied by 3 orders of magnitude [Jentzsch, K., Wirtz, A., Circolone, F., Drepper, T., Losi, A., Gärtner, W., Jaeger, K. E., and Krauss, U. (2009) Biochemistry 48, 10321-10333]. We now present evidence of the conservation of similar fast and slow-reverting "short" LOV proteins in different Pseudomonas species. Truncation studies conducted with PpSB1-LOV and PpSB2-LOV suggested that the short N- and C-terminal extensions outside of the LOV core domain are essential for the structural integrity and folding of the two proteins. While circular dichroism and solution nuclear magnetic resonance experiments verify that the two short C-terminal extensions of PpSB1-LOV and PpSB2-LOV form independently folding helical structures in solution, bioinformatic analyses imply the formation of coiled coils of the respective structural elements in the context of the dimeric full-length proteins. Given their prototypic architecture, conserved in most more complex LOV photoreceptor systems, "short" LOV proteins could represent ideally suited building blocks for the design of genetically encoded photoswitches (i.e., LOV-based optogenetic tools).


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Oxígeno/metabolismo , Proteínas Bacterianas/metabolismo , Mononucleótido de Flavina/metabolismo , Cinética , Luz , Optogenética , Oxígeno/química , Fotorreceptores Microbianos/genética , Fotorreceptores Microbianos/metabolismo , Estructura Terciaria de Proteína , Pseudomonas putida/metabolismo
8.
J Pept Sci ; 18(11): 691-5, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23001756

RESUMEN

The aggregation of the Aß plays a fundamental role in the pathology of AD. Recently, N-terminally modified Aß species, pE-Aß, have been described as major constituents of Aß deposits in the brains of AD patients. pE-Aß has an increased aggregation propensity and shows increased toxicity compared with Aß1-40 and Aß1-42. In the present work, high-resolution NMR spectroscopy was performed to study pE-Aß3-40 in aqueous TFE-containing solution. Two-dimensional TOCSY and NOESY experiments were performed. On the basis of NOE and chemical shift data, pE-Aß3-40 was shown to contain two helical regions formed by residues 14-22 and 30-36. This is similar as previously described for Aß1-40. However, the secondary chemical shift data indicate decreased helical propensity in pE-Aß3-40 when compared with Aß1-40 under exactly the same conditions. This is in agreement with the observation that pE-Aß3-40 shows a drastically increased tendency to form ß-sheet-rich structures under more physiologic conditions. Structural studies of pE-Aß are crucial for better understanding the structural basis of amyloid fibril formation in the brain during development of AD, especially because an increasing number of reports indicate a decisive role of pE-Aß for the pathogenesis of AD.


Asunto(s)
Péptidos beta-Amiloides/química , Espectroscopía de Resonancia Magnética , Ácido Pirrolidona Carboxílico/química , Enfermedad de Alzheimer/patología , Humanos , Isoformas de Proteínas/química
9.
Structure ; 20(10): 1778-87, 2012 Oct 10.
Artículo en Inglés | MEDLINE | ID: mdl-22959622

RESUMEN

Type 1 secretion systems (T1SS) catalyze the one step protein transport across the membranes of Gram-negative bacteria and are composed of an outer membrane protein, a membrane fusion protein and an ABC transporter. The ABC transporter consists of the canonical nucleotide binding and transmembrane domains. For the toxin hemolysin A (HlyA), the ABC transporter HlyB carries an additional, N-terminal domain sharing about 40% homology to C39 peptidases, but this "C39-like domain" (CLD) is suggested to feature another, yet unknown function. Our functional and structural analysis demonstrates that the CLD is essential for secretion and that it specifically interacts with the unfolded state of HlyA. We determined the nuclear magnetic resonance structure of the CLD as well as the substrate-binding region within the CLD. This mode of action, represents a mechanism within T1SS and answers the question, how a large and unfolded substrate is protected inside the cells during secretion.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Portadoras/química , Escherichia coli/metabolismo , Proteínas Hemolisinas/química , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Escherichia coli/aislamiento & purificación , Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/aislamiento & purificación , Proteínas Hemolisinas/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Triptófano/química
10.
Artículo en Inglés | MEDLINE | ID: mdl-21543878

RESUMEN

The ABC transporter haemolysin B (HlyB) from Escherichia coli is part of a type I secretion system that translocates a 110 kDa toxin in one step across both membranes of this Gram-negative bacterium in an ATP-dependent manner. Sequence analysis indicates that HlyB contains a C39 peptidase-like domain at its N-terminus. C39 domains are thiol-dependent peptidases that cleave their substrates after a GG motif. Interestingly, the catalytically invariant cysteine is replaced by a tyrosine in the C39-like domain of HlyB. Here, the overexpression, purification and crystallization of the isolated C39-like domain are described as a first step towards obtaining structural insights into this domain and eventually answering the question concerning the function of a degenerated C39 domain in the ABC transporter HlyB.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Proteínas Bacterianas/química , Proteínas Portadoras/química , Escherichia coli/química , Proteínas Hemolisinas/química , Replegamiento Proteico , Cristalización , Cristalografía por Rayos X , Multimerización de Proteína
11.
Proc Natl Acad Sci U S A ; 108(15): 6121-6, 2011 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-21430265

RESUMEN

Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels, are activated by binding of ligands to a domain (CNBD) located on the cytoplasmic side of the channel. The underlying mechanisms are not well understood. To elucidate the gating mechanism, structures of both the ligand-free and -bound CNBD are required. Several crystal structures of the CNBD from HCN2 and a bacterial CNG channel (MloK1) have been solved. However, for HCN2, the cAMP-free and -bound state did not reveal substantial structural rearrangements. For MloK1, structural information for the cAMP-free state has only been gained from mutant CNBDs. Moreover, in the crystal, the CNBD molecules form an interface between dimers, proposed to be important for allosteric channel gating. Here, we have determined the solution structure by NMR spectroscopy of the cAMP-free wild-type CNBD of MloK1. A comparison of the solution structure of cAMP-free and -bound states reveals large conformational rearrangement on ligand binding. The two structures provide insights on a unique set of conformational events that accompany gating within the ligand-binding site.


Asunto(s)
Alphaproteobacteria/metabolismo , AMP Cíclico/química , Canales Catiónicos Regulados por Nucleótidos Cíclicos/química , Cristalografía por Rayos X , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Mutación , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de Proteína
12.
Biomol NMR Assign ; 5(2): 199-201, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21347827

RESUMEN

ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins, which catalyze the translocation of molecules across biological membranes in an ATP-dependent manner. Despite the diversity in the transported substrates, they all share the same architecture, comprised of two transmembrane (TMD) and two nucleotide-binding domains (NBD). Members of the bacteriocin ABC transporter subfamily feature a special domain, belonging to the C39 (cystein protease family 39) peptidase protein family. These domains are assumed to cleave a C-terminal signal sequence from the protein or peptide substrate before or during the transport process. Although the C39 peptidase-like domain of the ABC transporter haemolysin B from E. coli shows no proteolytic activity, it is essential for the function of this transporter. In order to elucidate the contribution of the isolated C39 peptidase-like domain in the whole transport process, the backbone and side chain (1)H, (15)N and (13)C-NMR chemical shifts have been assigned.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Proteínas de Escherichia coli/química , Proteínas Hemolisinas/química , Resonancia Magnética Nuclear Biomolecular , Dominio Catalítico , Escherichia coli/enzimología , Isótopos/química
13.
Biomol NMR Assign ; 4(2): 147-50, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20449776

RESUMEN

Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels play crucial roles in neuronal excitability and signal transduction of sensory cells. These channels are activated by binding of cyclic nucleotides to their intracellular cyclic nucleotide-binding domain (CNBD). A comparison of the structures of wildtype ligand-free and ligand-bound CNBD is essential to elucidate the mechanism underlying nucleotide-dependent activation of CNBDs. We recently reported the solution structure of the Mesorhizobium loti K1 (MloK1) channel CNBD in complex with cAMP. We have now extended these studies and achieved nearly complete assignments of (1)H, (13)C and (15)N resonances of the nucleotide-free CNBD. A completely new assignment of the nucleotide-free wildtype CNBD was necessary due to the sizable chemical shift differences as compared to the cAMP bound CNBD and the slow exchange behaviour between both forms. Scattering of these chemical shift differences over the complete CNBD suggests that nucleotide binding induces significant overall conformational changes.


Asunto(s)
Canales Catiónicos Regulados por Nucleótidos Cíclicos/química , Resonancia Magnética Nuclear Biomolecular , Nucleótidos/metabolismo , Rhizobium/metabolismo , Estructura Terciaria de Proteína
14.
J Mol Biol ; 389(1): 58-73, 2009 May 29.
Artículo en Inglés | MEDLINE | ID: mdl-19362561

RESUMEN

Substrate-binding proteins or extracellular solute receptors (ESRs) are components of both ABC (ATP binding cassette) and TRAP-T (tripartite ATP-independent periplasmic transporter). The TRAP-T system UehABC from Silicibacter pomeroyi DSS-3 imports the compatible solutes ectoine and 5-hydroxyectoine as nutrients. UehA, the ESR of the UehABC operon, binds both ectoine and 5-hydroxyectoine with high affinity (K(d) values of 1.4+/-0.1 and 1.1+/-0.1 microM, respectively) and delivers them to the TRAP-T complex. The crystal structure of UehA in complex with ectoine was determined at 2.9-A resolution and revealed an overall fold common for all ESR proteins from TRAP systems determined so far. A comparison of the recently described structure of TeaA from Halomonas elongata and an ectoine-binding protein (EhuB) from an ABC transporter revealed a conserved ligand binding mode that involves both directed and cation-pi interactions. Furthermore, a comparison with other known TRAP-T ESRs revealed a helix that might act as a selectivity filter imposing restraints on the ESRs that fine-tune ligand recognition and binding and finally might determine the selection of the cognate substrate.


Asunto(s)
Aminoácidos Diaminos/química , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Sitios de Unión , Transporte Biológico , Cristalografía por Rayos X , Genoma Bacteriano , Halomonas/química , Cinética , Ligandos , Modelos Moleculares , Unión Proteica , Estructura Secundaria de Proteína , Rhodobacteraceae/enzimología , Rhodobacteraceae/genética , Especificidad por Sustrato
15.
J Bacteriol ; 190(16): 5663-71, 2008 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-18567662

RESUMEN

In the soil bacterium Bacillus subtilis, five transport systems work in concert to mediate the import of various compatible solutes that counteract the deleterious effects of increases in the osmolarity of the environment. Among these five systems, the ABC transporter OpuA, which catalyzes the import of glycine betaine and proline betaine, has been studied in detail in the past. Here, we demonstrate that OpuA is capable of importing the sulfobetaine dimethylsulfonioacetate (DMSA). Since OpuA is a classic ABC importer that relies on a substrate-binding protein priming the transporter with specificity and selectivity, we analyzed the OpuA-binding protein OpuAC by structural and mutational means with respect to DMSA binding. The determined crystal structure of OpuAC in complex with DMSA at a 2.8-A resolution and a detailed mutational analysis of these residues revealed a hierarchy within the amino acids participating in substrate binding. This finding is different from those for other binding proteins that recognize compatible solutes. Furthermore, important principles that enable OpuAC to specifically bind various compatible solutes were uncovered.


Asunto(s)
Bacillus subtilis/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Lipoproteínas/química , Lipoproteínas/metabolismo , Compuestos de Sulfonio/metabolismo , Sustitución de Aminoácidos/genética , Bacillus subtilis/genética , Bacillus subtilis/crecimiento & desarrollo , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Lipoproteínas/genética , Modelos Moleculares , Estructura Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Especificidad por Sustrato
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