RESUMEN

Elongin is a transcription elongation factor that stimulates the rate of elongation by suppressing transient pausing by RNA polymerase II at many sites along the DNA. It is heterotrimeric in mammals, consisting of elongins A, B and C subunits, and bears overall similarity to a class of E3 ubiquitin ligases known as SCF (Skp1-Cdc53 (cullin)-F-box) complexes. A subcomplex of elongins B and C is a target for negative regulation by the von Hippel-Lindau (VHL) tumor-suppressor protein. Elongin C from Saccharomyces cerevisiae, Elc1, exhibits high sequence similarity to mammalian elongin C. Using NMR spectroscopy we have determined the three-dimensional structure of Elc1 in complex with a human VHL peptide, VHL(157-171), representing the major Elc1 binding site. The bound VHL peptide is entirely helical. Elc1 utilizes two C-terminal helices and an intervening loop to form a binding groove that fits VHL(157-171). Chemical shift perturbation and dynamics analyses reveal that a global conformational change accompanies Elc1/VHL(157-171) complex formation. Moreover, the disappearance of conformational exchange phenomena on the microsecond to millisecond time scale within Elc1 upon VHL peptide binding suggests a role for slow internal motions in ligand recognition.

Asunto(s)

Ligasas , Proteínas/química , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Sitios de Unión , Elonguina , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Proteínas/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau , Levaduras/química

RESUMEN

Replication protein A (RPA) is a heterotrimeric, multi-functional protein that binds single-stranded DNA (ssDNA) and is essential for eukaryotic DNA metabolism. Using heteronuclear NMR methods we have investigated the domain interactions and ssDNA binding of a fragment from the 70 kDa subunit of human RPA (hRPA70). This fragment contains an N-terminal domain (NTD), which is important for hRPA70-protein interactions, connected to a ssDNA-binding domain (SSB1) by a flexible linker (hRPA70(1-326)). Correlation analysis of the amide (1)H and (15)N chemical shifts was used to compare the structure of the NTD and SSB1 in hRPA70(1-326) with two smaller fragments that corresponded to the individual domains. High correlation coefficients verified that the NTD and SSB1 maintained their structures in hRPA70(1-326), indicating weak interdomain coupling. Weak interdomain coupling was also suggested by a comparison of the transverse relaxation rates for hRPA70(1-326) and one of the smaller hRPA70 fragments containing the NTD and the flexible linker (hRPA70(1-168)). We also examined the structure of hRPA70(1-326) after addition of three different ssDNA substrates. Each of these substrates induced specific amide (1)H and/or (15)N chemical shift changes in both the NTD and SSB1. The NTD and SSB1 have similar topologies, leading to the possibility that ssDNA binding induced the chemical shift changes observed for the NTD. To test this hypothesis we monitored the amide (1)H and (15)N chemical shift changes of hRPA70(1-168) after addition of ssDNA. The same amide (1)H and (15)N chemical shift changes were observed for the NTD in hRPA70(1-168) and hRPA70(1-326). The NTD residues with the largest amide (1)H and/or (15)N chemical shift changes were localized to a basic cleft that is important for hRPA70-protein interactions. Based on this relationship, and other available data, we propose a model where binding between the NTD and ssDNA interferes with hRPA70-protein interactions.

Asunto(s)

ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Amidas/metabolismo , Secuencias de Aminoácidos , Secuencia Conservada , ADN de Cadena Simple/genética , Humanos , Cinética , Modelos Moleculares , Peso Molecular , Resonancia Magnética Nuclear Biomolecular , Docilidad , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína , Proteína de Replicación A , Rotación

RESUMEN

The binding of human immunodeficiency virus type 1 (HIV-1) Rev protein to its viral RNA target, stem-loop IIB (SLIIB) within the Rev Response element (RRE), mediates the export of singly-spliced and unspliced viral mRNA from the nucleus to the cytoplasm of infected cells; this Rev-mediated transport of viral RNA is absolutely required for the replication of infectious virus. To identify important features that influence the binding affinity and specificity of this Rev-RRE interaction, we have characterized the arginine side-chain dynamics of the Rev arginine-rich motif (ARM) while bound to a 34 nt RNA oligomer that corresponds to SLIIB. As the specificity of the Rev-RRE interaction varies with salt concentration, arginine side-chain dynamics were characterized at two different salt conditions. Following NMR measurements of (15)N spin relaxation parameters for the arginine (15)N(epsilon) nuclei, the dynamics of the corresponding N(epsilon)-H(epsilon) bond vectors were interpreted in terms of Lipari-Szabo model-free parameters using anisotropic expressions for the spectral density functions. Results from these analyses indicate that a number of arginine side-chains display a surprising degree of conformational freedom when bound to RNA, and that arginine residues having known importance for specific RRE recognition show striking differences in side-chain mobility. The (15)N relaxation measurements at different salt conditions suggest that the previously reported increase in Rev-RRE specificity at elevated salt concentrations is likely due to reduced affinity of non-specific Rev-RNA interactions. The observed dynamical behavior of the arginine side-chains at this protein-RNA interface likely plays an important role in the specificity and affinity of Rev-SLIIB complex formation.

Asunto(s)

Arginina/metabolismo , Productos del Gen rev/metabolismo , VIH-1/genética , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Anisotropía , Secuencia de Bases , Sitios de Unión , Transporte Biológico , Difusión , Productos del Gen rev/química , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligorribonucleótidos/química , Oligorribonucleótidos/genética , Oligorribonucleótidos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica/efectos de los fármacos , Conformación Proteica , ARN Viral/química , ARN Viral/genética , Proteínas de Unión al ARN/química , Sales (Química)/farmacología , Especificidad por Sustrato , Productos del Gen rev del Virus de la Inmunodeficiencia Humana

RESUMEN

Elongin is a transcription elongation factor that was first identified in mammalian systems and is composed of the three subunits, elongin A, B, and C. Sequence homologues of elongin A and elongin C, but not elongin B, were identified in the yeast genome. Neither yeast elongin A nor C sequence homologues was required for cell viability. The two gene products could be purified from yeast as a complex. A recombinant form of the complex, which could only be produced in bacteria if the gene products were co-expressed, was purified over several chromatographic steps. The complex did not stimulate transcription elongation by yeast RNA polymerase II. Using limited proteolysis, the N-terminal 144 residues of yeast elongin A were shown to be sufficient for interaction with yeast elongin C. The purified complex of yeast elongin C/elongin A(1-143) was analyzed using circular dichroism and nuclear magnetic spectroscopy. These studies revealed that yeast elongin A is unfolded but undergoes a dramatic modification of its structure in the presence of elongin C, and that elongin C forms a stable dimer in the absence of elongin A.

Asunto(s)

Proteínas Fúngicas/química , Saccharomyces cerevisiae/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Dicroismo Circular , Dimerización , Elonguina , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Factores de Transcripción/aislamiento & purificación , Transcripción Genética

RESUMEN

Elongin is a heterotrimeric transcription elongation factor composed of subunits A, B, and C in mammals. Elongin A and C are F-box-containing and SKP1 homologue proteins, respectively, and are therefore of interest for their potential roles in cell cycle-dependent proteolysis. Mammalian elongin C interacts with both elongin A and elongin B, as well as with the von Hippel-Lindau tumor suppressor protein VHL. To investigate the corresponding interactions in yeast, we have utilized NMR spectroscopy combined with ultracentrifugal sedimentation experiments to examine complexes of yeast elongin C (Elc1) with yeast elongin A (Ela1) and two peptides from homologous regions of Ela1 and human VHL. Elc1 alone is a homotetramer composed of subunits with a structured N-terminal region and a dynamically unstable C-terminal region. Binding of a peptide fragment of the Elc1-interaction domain of Ela1 or with a homologous peptide from VHL promotes folding of the C-terminal region of Elc1 into two regular helical structures and dissociates Elc1 into homodimers. Moreover, analysis of the complex of Elc1 with the full Elc1-interaction domain of Ela1 reveals that the Elc1 homodimer is dissociated to preferentially form an Ela1/Elc1 heterodimer. Thus, elongin C is found to oligomerize in solution and to undergo significant structural rearrangements upon binding of two different partner proteins. These results suggest a structural basis for the interaction of an F-box-containing protein with a SKP1 homologue and the modulation of this interaction by the tumor suppressor VHL.

Asunto(s)

Ligasas , Proteínas/química , Proteínas/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Secuencia de Aminoácidos , Animales , Elonguina , Genes Supresores de Tumor , Humanos , Enlace de Hidrógeno , Sustancias Macromoleculares , Mamíferos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Ultracentrifugación , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau

RESUMEN

BACKGROUND: The peptide segment surrounding residues Leu22 and Trp23 of the p53 transactivation domain plays a critical role in the transactivation activity of p53. This region binds basal transcriptional components such as the TATA-box binding protein associated factors TAFII40 and TAFII60 as well as the mdm-2 and adenovirus type 5 E1B 55 kDa oncoproteins. RESULTS: The structure of residues 14-28 of p53 was studied by nuclear magnetic resonance spectroscopy and found to prefer a two-beta-turn structure stabilized by a hydrophobic cluster consisting of residues known to be important for transactivation and binding to p53-binding proteins. A peptide segment in which Leu22 and Trp23 were replaced by Gln and Ser displays a random structure. CONCLUSIONS: This structural propensity observed in the wild-type p53 peptide is important for understanding the mechanism of transcriptional activation, because very few structural data are available on transactivation domains to date. These results should aid in the design of therapeutics that could competitively inhibit binding of p53 to the oncogene product mdm-2.

Asunto(s)

Conformación Proteica , Transactivadores/química , Proteína p53 Supresora de Tumor/química , Secuencia de Aminoácidos , Sitios de Unión , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Estructura Terciaria de Proteína , Soluciones , Temperatura

RESUMEN

The solution structure of the Cu(I) form of the rusticyanin from Thiobacillus ferrooxidans has been calculated from a total of 1979 distance and dihedral angle constraints derived from 1H, 13C and 15N NMR spectra. The structures reveal two beta-sheets, one of six strands and one of seven strands that are tightly packed in a beta-barrel or beta-sandwich arrangement, and a short helix that extends on the outside of one of the sheets to form a second hydrophobic core. The copper coordination sphere is composed of the standard type I ligands (His2CysMet) in a distorted tetrahedral arrangement. The copper-binding site is located within a hydrophobic region at one end of the molecule, surrounded by a number of aromatic rings and hydrophobic residues. This configuration probably contributes to the acid stability of the copper site, since close association of the aromatic rings with the histidine ligands would sterically hinder their dissociation from the copper. An electrostatic analysis based on a comparison of the structures of rusticyanin and French bean plastocyanin shows that factors determining the high redox potential of rusticyanin include contributions from charged side-chains and from the disposition of backbone peptide dipoles, particularly in the 81 to 86 region of the sequence and the ligand cysteine residue. These interactions should also contribute to the acid stability by inhibiting protonation of His143.

Asunto(s)

Azurina/análogos & derivados , Thiobacillus/química , Azurina/química , Azurina/metabolismo , Sitios de Unión , Cobre/metabolismo , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Oxidación-Reducción , Soluciones , Difracción de Rayos X

RESUMEN

An alkaline phosphatase assay was used to determine the dissociation constants (KD) of the lac repressor N-terminal 56 amino acid fragment of the wild type and of a Y7I mutant complexed to 22 base pair (bp) wild-type and mutant symmetrical operator sequences. KD's in 0.35 M monovalent salt ranged from 5.4 X 10(-8) M for the wild-type repressor.wild-type operator complex to approximately > 1 X 10(-6) M for the wild-type repressor.nonspecific DNA complex. Mutant operators O2 (G5 --> A5 and G16 --> T16) and O4 (G5 --> C5 and C16 --> G16) bind nearly as tightly as the wild-type headpiece, while mutant O3 (A8 --> T8 and T13 --> A13) binds over 5-fold poorer. Operators O1, O2, and O4 bind ca. 10-fold poorer to the Y7I mutant headpiece. Operator O3 binds 2-fold poorer to the mutant headpiece. The temperature and salt dependence on the dissociation constants of wild-type headpiece binding to 22-bp operator support the conclusion that the headpiece contains the major DNA recognition portion of the protein and that electrostatics plays as important a role in the binding of operator to headpiece as it does in the whole repressor. The 31P NMR spectra of shortened 14-bp wild-type and mutant symmetrical operators bound to the N-terminal 56-residue headpiece of the Y7I mutant repressor were compared to the spectra of the same operator bound to the wild-type repressor headpiece. These results are consistent with a recent proposal [Karslake, C., Botuyan, M. V., & Gorenstein, D. G. (1992) Biochemistry 31, 1849-1858] that specific, tight-binding operator.protein complexes retain the inherent phosphate ester conformational flexibility of the operator itself, whereas the phosphate esters are conformationally restricted in the weak-binding operator-protein complexes. This retention of backbone torsional freedom in tight complexes is entropically favorable and provides a mechanism for protein discrimination of different operator binding sites.

Asunto(s)

Proteínas de Unión al ADN/química , ADN/metabolismo , Regiones Operadoras Genéticas , Proteínas Represoras/química , Fosfatasa Alcalina/metabolismo , Secuencia de Bases , Proteínas de Unión al ADN/metabolismo , Ésteres/química , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Mutación , Isótopos de Fósforo , Proteínas Represoras/metabolismo , Cloruro de Sodio/química , Temperatura

RESUMEN

The 31P NMR spectra of various 14-base-pair lac operators bound to both wild-type and mutant lac repressor headpiece proteins were analyzed to provide information on the backbone conformation in the complexes. The 31P NMR spectrum of a wild-type symmetrical operator, d(TGTGAGCGCTCACA)2, bound to the N-terminal 56-residue headpiece fragment of a Y7I mutant repressor was nearly identical to the spectrum of the same operator bound to the wild-type repressor headpiece. In contrast, the 31P NMR spectrum of the mutant operator, d(TATAGAGCGCTCATA)2, wild-type headpiece complex was significantly perturbed relative to the wild-type repressor-operator complex. The 31P chemical shifts of the phosphates of a second mutant operator, d(TGTGTGCGCACACA)2, showed small but specific changes upon complexation with either the wild-type or mutant headpiece. The 31P chemical shifts of the phosphates of a third mutant operator, d(TCTGAGCGCTCAGA)2, showed no perturbations upon addition of the wild-type headpiece. The 31P NMR results provide further evidence for predominant recognition of the 5'-strand of the 5'-TGTGA/3'-ACACT binding site in a 2:1 protein to headpiece complex. It is proposed that specific, strong-binding operator-protein complexes retain the inherent phosphate ester conformational flexibility of the operator itself, whereas the phosphate esters are conformationally restricted in the weak-binding operator-protein complexes. This retention of backbone torsional freedom in strong complexes is entropically favorable and provides a new (and speculative) mechanism for protein discrimination of different operator binding sites. It demonstrates the potential importance of phosphate geometry and flexibility on protein recognition and binding.

Asunto(s)

ADN/metabolismo , Ésteres/química , Operón Lac , Espectroscopía de Resonancia Magnética , Oligodesoxirribonucleótidos/metabolismo , Fosfatos/química , Proteínas Represoras/metabolismo , Secuencia de Bases , Sitios de Unión , Escherichia coli/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos/química , Plásmidos , Proteínas Represoras/química