RESUMEN
The thermodynamics of secondary p53 binding sites on MDM2 and MDMX were evaluated using p53 peptides containing residues 16-29, 17-35, and 1-73. All the peptides had large, negative heat capacity (ΔCp), consistent with the burial of p53 residues F19, W23, and L26 in the primary binding sites of MDM2 and MDMX. MDMX has a higher affinity and more negative ΔCp than MDM2 for p5317-35, which is due to MDMX stabilization and not additional interactions with the secondary binding site. ΔCp measurements show binding to the secondary site is inhibited by the disordered tails of MDM2 for WT p53 but not a more helical mutant where proline 27 is changed to alanine. This result is supported by all-atom molecular dynamics simulations showing that p53 residues 30-35 turn away from the disordered tails of MDM2 in P27A17-35 and make direct contact with this region in p5317-35. Molecular dynamics simulations also suggest that an intramolecular methionine-aromatic motif found in both MDM2 and MDMX structurally adapts to support multiple p53 binding modes with the secondary site. ΔCp measurements also show that tighter binding of the P27A mutant to MDM2 and MDMX is due to increased helicity, which reduces the energetic penalty associated with coupled folding and binding. Our results will facilitate the design of selective p53 inhibitors for MDM2 and MDMX.
Asunto(s)
Proteínas de Ciclo Celular , Simulación de Dinámica Molecular , Proteínas Proto-Oncogénicas c-mdm2 , Proteínas Proto-Oncogénicas , Termodinámica , Proteína p53 Supresora de Tumor , Humanos , Sitios de Unión , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Unión Proteica , Conformación Proteica , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-mdm2/química , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Phosphomannomutase/phosphoglucomutase contributes to the infectivity of Pseudomonas aeruginosa, retains and reorients its intermediate by 180°, and rotates domain 4 to close the deep catalytic cleft. Nuclear magnetic resonance (NMR) spectra of the backbone of wild-type and S108C-inactivated enzymes were assigned to at least 90%. (13)C secondary chemical shifts report excellent agreement of solution and crystallographic structure over the 14 α-helices, C-capping motifs, and 20 of the 22 ß-strands. Major and minor NMR peaks implicate substates affecting 28% of assigned residues. These can be attributed to the phosphorylation state and possibly to conformational interconversions. The S108C substitution of the phosphoryl donor and acceptor slowed transformation of the glucose 1-phosphate substrate by impairing k(cat). Addition of the glucose 1,6-bisphosphate intermediate accelerated this reaction by 2-3 orders of magnitude, somewhat bypassing the defect and apparently relieving substrate inhibition. The S108C mutation perturbs the NMR spectra and electron density map around the catalytic cleft while preserving the secondary structure in solution. Diminished peak heights and faster (15)N relaxation suggest line broadening and millisecond fluctuations within four loops that can contact phosphosugars. (15)N NMR relaxation and peak heights suggest that domain 4 reorients slightly faster in solution than domains 1-3, and with a different principal axis of diffusion. This adds to the crystallographic evidence of domain 4 rotations in the enzyme, which were previously suggested to couple to reorientation of the intermediate, substrate binding, and product release.
Asunto(s)
Fosfotransferasas (Fosfomutasas)/química , Fosfotransferasas (Fosfomutasas)/genética , Dominio Catalítico/genética , Cristalografía por Rayos X , Resonancia Magnética Nuclear Biomolecular , Fosfoglucomutasa/química , Fosfoglucomutasa/genética , Fosforilación/genética , Fosfotransferasas (Fosfomutasas)/metabolismo , Unión Proteica/genética , Transporte de Proteínas/genética , Pseudomonas aeruginosa/enzimología , Especificidad por Sustrato/genéticaRESUMEN
Autoinhibition of p53 binding to MDMX requires two short-linear motifs (SLiMs) containing adjacent tryptophan (WW) and tryptophan-phenylalanine (WF) residues. NMR spectroscopy was used to show the WW and WF motifs directly compete for the p53 binding site on MDMX and circular dichroism spectroscopy was used to show the WW motif becomes helical when it is bound to the p53 binding domain (p53BD) of MDMX. Binding studies using isothermal titration calorimetry showed the WW motif is a stronger inhibitor of p53 binding than the WF motif when they are both tethered to p53BD by the natural disordered linker. We also investigated how the WW and WF motifs interact with the DNA binding domain (DBD) of p53. Both motifs bind independently to similar sites on DBD that overlap the DNA binding site. Taken together our work defines a model for complex formation between MDMX and p53 where a pair of disordered SLiMs bind overlapping sites on both proteins.
Asunto(s)
Proteínas Proto-Oncogénicas c-mdm2 , Proteína p53 Supresora de Tumor , Fenilalanina/química , Unión Proteica , Proteínas Proto-Oncogénicas c-mdm2/química , Triptófano/química , Proteína p53 Supresora de Tumor/química , Secuencias de Aminoácidos , Dominios Proteicos , HumanosRESUMEN
Aqueous solubilities of many drugs in current clinical use are very low, necessitating formulations that often present problems for parenteral administration, including toxicities due to the excipients used. Recognizing that pharmacologically active compounds frequently possess amines, we asked whether pyridoxal phosphate (PLP), an inoccuous, water-soluble vitamin, could be utilized to form prodrug-like complexes via the formation of imine or iminium adducts and whether the vitamin would impart solubilizing properties to such complexes. Direct spectroscopic and crystallographic data obtained using model primary and secondary amines showed that PLP forms stable imine adducts with primary amines under entirely aqueous conditions and at physiologic pH, while no reaction was observed for secondary amines; the basis of the exceptional stability appears to be a consequence of favorable H-bond interactions of the imine nitrogen with the 5-OH group of PLP. Amphotericin B and nystatin in their native forms display marked aqueous insolubility and possess lone primary amines. We were able to utilize PLP in achieving excellent solubilization of both of these antifungal agents, surpassing aqueous solubilities of 100 mg/mL. In in vitro bioassays, both polyenes in their PLP-adducted form display attenuated antifungal potencies which are attributable to "prodrug-like" complexes. These results point to the utility of excipient-free, entirely aqueous formulations of amphotericin B for parenteral use, and may also be extended to other primary amine-bearing compounds exhibiting poor aqueous solubility.
Asunto(s)
Anfotericina B/química , Nistatina/química , Fosfato de Piridoxal/química , Profármacos/química , SolubilidadRESUMEN
The autosomal-dominant pleiotropic disorder called oculodentodigital dysplasia (ODDD) is caused by mutations in the gap junction protein Cx43. Of the 73 mutations identified to date, over one-third are localized in the cytoplasmic loop (Cx43CL) domain. Here, we determined the mechanism by which three ODDD mutations (M147T, R148Q, and T154A), all of which localize within the predicted 1-5-10 calmodulin-binding motif of the Cx43CL, manifest the disease. Nuclear magnetic resonance (NMR) and circular dichroism revealed that the three ODDD mutations had little-to-no effect on the ability of the Cx43CL to form α-helical structure as well as bind calmodulin. Combination of microscopy and a dye-transfer assay uncovered these mutations increased the intracellular level of Cx43 and those that trafficked to the plasma membrane did not form functional channels. NMR also identify that CaM can directly interact with the Cx43CT domain. The Cx43CT residues involved in the CaM interaction overlap with tyrosines phosphorylated by Pyk2 and Src. In vitro and in cyto data provide evidence that the importance of the CaM interaction with the Cx43CT may lie in restricting Pyk2 and Src phosphorylation, and their subsequent downstream effects.
Asunto(s)
Calmodulina/genética , Conexina 43/genética , Anomalías Craneofaciales/genética , Anomalías del Ojo/genética , Deformidades Congénitas del Pie/genética , Sindactilia/genética , Anomalías Dentarias/genética , Calmodulina/ultraestructura , Movimiento Celular/genética , Conexina 43/ultraestructura , Anomalías Craneofaciales/patología , Citoplasma/genética , Anomalías del Ojo/patología , Quinasa 2 de Adhesión Focal/genética , Deformidades Congénitas del Pie/patología , Uniones Comunicantes/genética , Células HeLa , Humanos , Mutación con Pérdida de Función/genética , Unión Proteica , Conformación Proteica en Hélice alfa , Transporte de Proteínas/genética , Sindactilia/patología , Anomalías Dentarias/patologíaRESUMEN
The lanthanides (Ln3+), or rare earth elements, have proven to be useful tools for biomolecular NMR, X-ray crystallographic, and fluorescence analyses due to their unique 4f orbitals. However, their utility in biological applications has been limited because site-specific incorporation of a chelating element is required to ensure efficient binding of the free Ln3+ ion. Additionally, current Ln3+ chelator syntheses complicate efforts to directly incorporate Ln3+ chelators into proteins as the multi-step processes and a reliance on organic solvents promote protein denaturation and aggregation which are generally incompatible with direct incorporation into the protein of interest. To overcome these limitations, herein we describe a two-step aqueous synthesis of a small molecule lanthanide chelating agent amenable to site-specific incorporation into a protein using copper-free click chemistry with unnatural amino acids. The bioconjugate combines a diethylenetriaminepentaacetic acid (DTPA) chelating moiety with a clickable dibenzylcyclooctyne-amine (DBCO-amine) to facilitate the reaction with an azide containing unnatural amino acid. Incorporating the DBCO-amine avoids the use of the cytotoxic Cu2+ ion as a catalyst. The clickable lanthanide chelator (CLC) reagent reacted readily with p-azidophenylalanine (paF) without the need of a copper catalyst, thereby demonstrating proof-of-concept. Implementation of the orthogonal click chemistry reaction has the added advantage that the chelator can be used directly in a protein labeling reaction, without the need of extensive purification. Given the inherent advantages of Cu2+-free click chemistry, aqueous synthesis, and facile labeling, we believe that the CLC will find abundant use in both structural and biophysical studies of proteins and their complexes.
Asunto(s)
Quelantes/síntesis química , Elementos de la Serie de los Lantanoides/química , Bibliotecas de Moléculas Pequeñas/síntesis química , Quelantes/química , Química Clic , Cobre/química , Hierro/química , Ácido Pentético/química , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Many pathogens such as Shigella and other bacteria assemble the typeâ III secretion system (T3SS) nanoinjector to inject virulence proteins into their target cells to cause infectious diseases in humans. The rise of drug resistance among pathogens that rely on the T3SS for infectivity, plus the dearth of new antibiotics require alternative strategies in developing new antibiotics. The Shigella T3SS tip protein IpaD is an attractive target for developing anti-infectives because of its essential role in virulence and its exposure on the bacterial surface. Currently, the only known small molecules that bind to IpaD are bile salt sterols. In this study we identified four new small-molecule scaffolds that bind to IpaD, based on the methylquinoline, pyrrolidine-aniline, hydroxyindole, and morpholinoaniline scaffolds. NMR mapping revealed potential hotspots in IpaD for binding small molecules. These scaffolds can be used as building blocks in developing small-molecule inhibitors of IpaD that could lead to new anti-infectives.
Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Shigella/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismo , Compuestos de Anilina/química , Compuestos de Anilina/metabolismo , Antígenos Bacterianos/genética , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Espectroscopía de Resonancia Magnética , Unión Proteica , Quinolinas/química , Quinolinas/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Bibliotecas de Moléculas Pequeñas/química , Resonancia por Plasmón de Superficie , Sistemas de Secreción Tipo IIIRESUMEN
The goal of this work is to probe the interaction between cyclic cHAVc3 peptide and the EC1 domain of human E-cadherin protein. Cyclic cHAVc3 peptide (cyclo(1,6)Ac-CSHAVC-NH2) binds to the EC1 domain as shown by chemical shift perturbations in the 2D 1H,-15N-HSQC NMR spectrum. The molecular dynamics (MD) simulations of the EC1 domain showed folding of the C-terminal tail region into the main head region of the EC1 domain. For cHAVc3 peptide, replica exchange molecular dynamics (REMD) simulations generated five structural clusters of cHAVc3 peptide. Representative structures of cHAVc3 and the EC1 structure from MD simulations were used in molecular docking experiments with NMR constraints to determine the binding site of the peptide on EC1. The results suggest that cHAVc3 binds to EC1 around residues Y36, S37, I38, I53, F77, S78, H79, and I94. The dissociation constants (Kd values) of cHAVc3 peptide to EC1 were estimated using the NMR chemical shifts data and the estimated Kds are in the range of .5 × 10-5-7.0 × 10-5 M.
Asunto(s)
Cadherinas/química , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Péptidos Cíclicos/química , Dominios y Motivos de Interacción de Proteínas , Sitios de Unión , Cadherinas/metabolismo , Enlace de Hidrógeno , Péptidos Cíclicos/metabolismo , Unión ProteicaRESUMEN
Many Gram-negative bacteria require the typeâ III secretion system (T3SS) to cause infectious diseases in humans. A looming public health problem is that all bacterial pathogens that require the T3SS to cause infectious diseases in humans have developed multidrug resistance to current antibiotics. The T3SS is an attractive target for the development of new antibiotics because of its critical role in virulence. An initial step in developing anti-T3SS-based therapeutics is the identification of small molecules that can bind to T3SS proteins. Currently, the only small molecules that are known to bind to the Salmonella T3SS proteins SipD and SipB are bile salts (to SipD) and sphingolipids and cholesterol (to SipB). Herein we report the results of a surface plasmon resonance screen of 288 compounds wherein the binding of 4-morpholinoaniline to SipD, 3-indoleacetic acid to SipB, and 5-hydroxyindole to both SipD and SipB were identified. We also identified by NMR the SipD surfaces involved in binding. These three compounds represent a new class of molecules that can bind to T3SS tip (SipD) and translocon (SipB) proteins that could find use in future drug design.
Asunto(s)
Compuestos de Anilina/metabolismo , Proteínas Bacterianas/metabolismo , Ácidos Indolacéticos/metabolismo , Indoles/metabolismo , Salmonella/metabolismo , Sistemas de Secreción Tipo III/metabolismo , Compuestos de Anilina/química , Antígenos Bacterianos/química , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/química , Ácidos Indolacéticos/química , Indoles/química , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Resonancia por Plasmón de Superficie , Sistemas de Secreción Tipo III/químicaRESUMEN
Protein-protein interactions represent an exciting and challenging target class for therapeutic intervention using small molecules. Protein interaction sites are often devoid of the deep surface pockets presented by "traditional" drug targets, and crystal structures reveal that inhibitors typically engage these sites using very shallow binding modes. As a consequence, modern virtual screening tools developed to identify inhibitors of traditional drug targets do not perform as well when they are instead deployed at protein interaction sites. To address the need for novel inhibitors of important protein interactions, here we introduce an alternate docking strategy specifically designed for this regime. Our method, termed DARC (Docking Approach using Ray-Casting), matches the topography of a surface pocket "observed" from within the protein to the topography "observed" when viewing a potential ligand from the same vantage point. We applied DARC to carry out a virtual screen against the protein interaction site of human antiapoptotic protein Mcl-1 and found that four of the top-scoring 21 compounds showed clear inhibition in a biochemical assay. The Ki values for these compounds ranged from 1.2 to 21 µM, and each had ligand efficiency comparable to promising small-molecule inhibitors of other protein-protein interactions. These hit compounds do not resemble the natural (protein) binding partner of Mcl-1, nor do they resemble any known inhibitors of Mcl-1. Our results thus demonstrate the utility of DARC for identifying novel inhibitors of protein-protein interactions.
Asunto(s)
Proteína 1 de la Secuencia de Leucemia de Células Mieloides/química , Humanos , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica , Propiedades de SuperficieRESUMEN
We have recently identified a Toll-like receptor (TLR4)-interacting SPA4 peptide encoding amino acids: GDFRYSDGTPVNYTNWYRGE, a shorter region of human surfactant protein-A (SP-A). The SPA4 peptide suppressed lipopolysaccharide-induced inflammation (JPET 2011, Innate Immun 2013). In this report, we examined the structure of synthetic SPA4 peptide in solution by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. The CD analysis revealed that the SPA4 peptide is composed of â¼35% beta sheet and <5% alpha helix. We used solution NMR to solve the structure of the SPA4 peptide. We calculated NMR structures using Nuclear Overhauser Enhancement (NOE) distance restraints. The superposition of the low energy structures indicated that the central 6-14 amino acids "SDGTPVNYT" of the 20mer SPA4 peptide form a turn, and amino acids on either side (GDFRY and NWYRGE) conform to flexible arms. Furthermore, thermal denaturation experiments demonstrated the structural flexibility of the peptide. The NMR structures of the SPA4 peptide align well with the homologous region within the available structure of rat SP-A and a computationally-docked model of SP-A-TLR4-MD2 protein complex. Together, our results support the structural adaptability of SPA4 peptide for binding to TLR4.
RESUMEN
The Extracellular 1 (EC1) domain of E-cadherin has been shown to be important for cadherin-cadherin homophilic interactions. Cadherins are responsible for calcium-mediated cell-cell adhesion located at the adherens junction of the biological barriers (i.e., intestinal mucosa and the blood-brain barrier (BBB)). Cadherin peptides can modulate cadherin interactions to improve drug delivery through the BBB. However, the mechanism of modulating the E-cadherin interactions by cadherin peptides has not been fully elucidated. To provide a basis for subsequent examination of the structure and peptide-binding properties of the EC1 domain of human E-cadherin using solution NMR spectroscopy, the (1)H, (13)C and (15)N backbone resonance of the uniformly labeled-EC1 were assigned and the secondary structure was determined based on the chemical shift values. These resonance assignments are essential for assessing protein-ligand interactions and are reported here.
Asunto(s)
Cadherinas/química , Resonancia Magnética Nuclear Biomolecular , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Estructura Terciaria de ProteínaRESUMEN
Musashi-1 (MSI1) is an RNA-binding protein that acts as a translation activator or repressor of target mRNAs. The best-characterized MSI1 target is Numb mRNA, whose encoded protein negatively regulates Notch signaling. Additional MSI1 targets include the mRNAs for the tumor suppressor protein APC that regulates Wnt signaling and the cyclin-dependent kinase inhibitor P21(WAF-1). We hypothesized that increased expression of NUMB, P21 and APC, through inhibition of MSI1 RNA-binding activity might be an effective way to simultaneously downregulate Wnt and Notch signaling, thus blocking the growth of a broad range of cancer cells. We used a fluorescence polarization assay to screen for small molecules that disrupt the binding of MSI1 to its consensus RNA binding site. One of the top hits was (-)-gossypol (Ki = 476 ± 273 nM), a natural product from cottonseed, known to have potent anti-tumor activity and which has recently completed Phase IIb clinical trials for prostate cancer. Surface plasmon resonance and nuclear magnetic resonance studies demonstrate a direct interaction of (-)-gossypol with the RNA binding pocket of MSI1. We further showed that (-)-gossypol reduces Notch/Wnt signaling in several colon cancer cell lines having high levels of MSI1, with reduced SURVIVIN expression and increased apoptosis/autophagy. Finally, we showed that orally administered (-)-gossypol inhibits colon cancer growth in a mouse xenograft model. Our study identifies (-)-gossypol as a potential small molecule inhibitor of MSI1-RNA interaction, and suggests that inhibition of MSI1's RNA binding activity may be an effective anti-cancer strategy.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/patología , Gosipol/farmacología , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas de Unión al ARN/efectos de los fármacos , Animales , Línea Celular , Línea Celular Tumoral , Neoplasias del Colon/metabolismo , Femenino , Humanos , Ratones , Ratones Desnudos , Proteínas del Tejido Nervioso/metabolismo , Unión Proteica , Proteínas de Unión al ARN/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
ChxR is an atypical two-component signal transduction response regulator (RR) of the OmpR/PhoB subfamily encoded by the obligate intracellular bacterial pathogen Chlamydia trachomatis. Despite structural homology within both receiver and effector domains to prototypical subfamily members, ChxR does not require phosphorylation for dimer formation, DNA binding or transcriptional activation. Thus, we hypothesized that ChxR is in a conformation optimal for DNA binding with limited interdomain interactions. To address this hypothesis, the NMR solution structure of the ChxR effector domain was determined and used in combination with the previously reported ChxR receiver domain structure to generate a full-length dimer model based upon SAXS analysis. Small-angle scattering of ChxR supported a dimer with minimal interdomain interactions and effector domains in a conformation that appears to require only subtle reorientation for optimal major/minor groove DNA interactions. SAXS modeling also supported that the effector domains were in a head-to-tail conformation, consistent with ChxR recognizing tandem DNA repeats. The effector domain structure was leveraged to identify key residues that were critical for maintaining protein - nucleic acid interactions. In combination with prior analysis of the essential location of specific nucleotides for ChxR recognition of DNA, a model of the full-length ChxR dimer bound to its cognate cis-acting element was generated.
Asunto(s)
Proteínas Bacterianas/química , Chlamydia trachomatis/metabolismo , ADN Bacteriano/química , Regulación Bacteriana de la Expresión Génica , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Chlamydia trachomatis/genética , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Secuencias Repetidas en Tándem , TermodinámicaRESUMEN
Norovirus protease is an essential enzyme for proteolytic maturation of norovirus nonstructural proteins and has been implicated as a potential target for antiviral drug development. Although X-ray structural studies of the protease give us wealth of structural information including interactions of the protease with its substrate and dimeric overall structure, the role of protein dynamics in the substrate recognition and the biological relevance of the protease dimer remain unclear. Here we determined the solution NMR structure of the 3C-like protease from Norwalk virus (NV 3CLpro), a prototype strain of norovirus, and analyzed its backbone dynamics and hydrodynamic behavior in solution. ¹5N spin relaxation and analytical ultracentrifugation analyses demonstrate that NV 3CLpro is predominantly a monomer in solution. Solution structure of NV 3CLpro shows significant structural variation in C-terminal domain compared with crystal structures and among lower energy structure ensembles. Also, ¹5N spin relaxation and Carr-Purcell-Meiboom-Gill (CPMG)-based relaxation dispersion analyses reveal the dynamic properties of residues in the C-terminal domain over a wide range of timescales. In particular, the long loop spanning residues T123-G133 show fast motion (ps-ns), and the residues in the bII-cII region forming the large hydrophobic pocket (S2 site) undergo conformational exchanges on slower timescales (µs-ms), suggesting their important role in substrate recognition.
Asunto(s)
Cisteína Endopeptidasas/química , Norovirus/enzimología , Proteínas Virales/química , Sitios de Unión , Biocatálisis , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Dimerización , Hidrodinámica , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidad , Especificidad por Sustrato , Ultracentrifugación , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Norovirus protease cleaves the virus-encoded polyprotein into six mature nonstructural proteins, presenting itself as an essential enzyme for the viral replication as well as an attractive target for the antiviral drug development. A deeper understanding of the structural mechanism of the protease-substrates/inhibitors interactions by means of solution NMR methods would facilitate a rational design of the virus protease inhibitor. We here report the backbone and side-chain resonance assignment of the protease from Norwalk virus, which is the prototype strain of norovirus. The assignment data has been deposited in the BMRB database under the accession number 17523.
Asunto(s)
Virus Norwalk/enzimología , Resonancia Magnética Nuclear Biomolecular , Péptido Hidrolasas/química , Antivirales/química , Antivirales/farmacología , Descubrimiento de Drogas , Virus Norwalk/efectos de los fármacos , Péptido Hidrolasas/metabolismo , Relación Estructura-ActividadRESUMEN
Domain 4 of the anthrax protective antigen (PA) plays a key role in cellular receptor recognition as well as in pH-dependent pore formation. We present here the 1.95 A crystal structure of domain 4, which adopts a fold that is identical to that observed in the full-length protein. We have also investigated the structural properties of the isolated domain 4 as a function of pH, as well as the pH-dependence on binding to the von Willebrand factor A domain of capillary morphogenesis protein 2 (CMG2). Our results provide evidence that the isolated domain 4 maintains structure and interactions with CMG2 at pH 5, a pH that is known to cause release of the receptor on conversion of the heptameric prepore (PA(63))(7) to a membrane-spanning pore. Our results suggest that receptor release is not driven solely by a pH-induced unfolding of domain 4.
Asunto(s)
Antígenos Bacterianos/metabolismo , Toxinas Bacterianas/metabolismo , Proteínas de la Membrana/metabolismo , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Cromatografía en Gel , Dicroismo Circular , Humanos , Concentración de Iones de Hidrógeno , Proteínas de la Membrana/química , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Estabilidad Proteica , Estructura Terciaria de Proteína , Receptores de Péptidos , Espectrometría de Fluorescencia , Temperatura , TermodinámicaRESUMEN
PR39, a naturally occurring and cell-permeable proline- and arginine-rich peptide, blocks the degradation of inhibitor of nuclear factor kappaB (IkappaBalpha), thereby attenuating inflammation. It is a noncompetitive and reversible inhibitor of 20S proteasome. To identify its basis of action, we used solution NMR spectroscopy and mutational analyses of the active fragment, PR11, which identified amino acids required for human 20S proteasome inhibiting activity. We then examined PR11-mediated changes in the expression of nuclear factor kappaB-dependent genes in situ. The results provide prerequisites for proteasome inhibition by proline- and arginine-rich peptides, providing a powerful new tool to investigate inflammatory processes. These findings offer new leads in developing drugs to treat heart diseases or stroke.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Inhibidores de Proteasoma , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos/química , Células Cultivadas , Quimotripsina/antagonistas & inhibidores , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/farmacología , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Transcription enhancer factor 1 is essential for cardiac, skeletal, and smooth muscle development and uses its N-terminal TEA domain (TEAD) to bind M-CAT elements. Here, we present the first structure of TEAD and show that it is a three-helix bundle with a homeodomain fold. Structural data reveal how TEAD binds DNA. Using structure-function correlations, we find that the L1 loop is essential for cooperative loading of TEAD molecules on to tandemly duplicated M-CAT sites. Furthermore, using a microarray chip-based assay, we establish that known binding sites of the full-length protein are only a subset of DNA elements recognized by TEAD. Our results provide a model for understanding the regulation of genome-wide gene expression during development by TEA/ATTS family of transcription factors.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN/genética , Proteínas de Unión al ADN/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Filogenia , Unión Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Factores de Transcripción/genéticaRESUMEN
The C-terminus of calmodulin (CaM) functions as a sensor of oxidative stress, with oxidation of methionine 144 and 145 inducing a nonproductive association of the oxidized CaM with the plasma membrane Ca(2+)-ATPase (PMCA) and other target proteins to downregulate cellular metabolism. To better understand the structural underpinnings and mechanism of this switch, we have engineered a CaM mutant (CaM-L7) that permits the site-specific oxidation of M144 and M145, and we have used NMR spectroscopy to identify structural changes in CaM and CaM-L7 and changes in the interactions between CaM-L7 and the CaM-binding sequence of the PMCA (C28W) due to methionine oxidation. In CaM and CaM-L7, methionine oxidation results in nominal secondary structural changes, but chemical shift changes and line broadening in NMR spectra indicate significant tertiary structural changes. For CaM-L7 bound to C28W, main chain and side chain chemical shift perturbations indicate that oxidation of M144 and M145 leads to large tertiary structural changes in the C-terminal hydrophobic pocket involving residues that comprise the interface with C28W. Smaller changes in the N-terminal domain also involving residues that interact with C28W are observed, as are changes in the central linker region. At the C-terminal helix, (1)H(alpha), (13)C(alpha), and (13)CO chemical shift changes indicate decreased helical character, with a complete loss of helicity for M144 and M145. Using (13)C-filtered, (13)C-edited NMR experiments, dramatic changes in intermolecular contacts between residues in the C-terminal domain of CaM-L7 and C28W accompany oxidation of M144 and M145, with an essentially complete loss of contacts between C28W and M144 and M145. We propose that the inability of CaM to fully activate the PMCA after methionine oxidation originates in a reduced helical propensity for M144 and M145, and results primarily from a global rearrangement of the tertiary structure of the C-terminal globular domain that substantially alters the interaction of this domain with the PMCA.