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1.
Pakuła S Orłowski M Rymarczyk G Krusiński T Jakób M Zoglowek A Ożyhar A Dobryszycki P 《Journal of biomolecular structure & dynamics》2012,30(4):379-393
The ecdysone receptor (EcR) and the ultraspiracle protein (Usp) form the functional receptor for ecdysteroids that initiates metamorphosis in insects. The Usp and EcR DNA-binding domains (UspDBD and EcRDBD, respectively) form a heterodimer on the natural pseudopalindromic element from the hsp27 gene promoter. The conformational changes in the protein-DNA during the formation of the UspDBD-EcRDBD-hsp27 complex were analyzed. Recombined UspDBD and EcRDBD proteins were purified and fluorescein labeled (FL) using the intein method at the C-ends of both proteins. The changes in the distances from the respective C-ends of EcRDBD and/or UspDBD to the 5'- and/or 3'-end of the response element were measured using fluorescence resonance energy transfer (FRET) methodology. The binding of EcRDBD induced a strong conformational change in UspDBD and caused the C-terminal fragment of the UspDBD molecule to move away from both ends of the regulatory element. UspDBD also induced a significant conformational change in the EcRDBD molecule. The EcRDBD C-terminus moved away from the 5'-end of the regulatory element and moved close to the 3'-end. An analysis was also done on the effect that DHR38DBD, the Drosophila ortholog of the mammalian NGFI-B, had on the interaction of UspDBD and EcRDBD with hsp27. FRET analysis demonstrated that hsp27 bending was induced by DHR38DBD. Fluorescence data revealed that hsp27 had a shorter end-to-end distance both in the presence of EcRDBD as well as in the presence of EcRDBD together with DHR38DBD, with DNA bend angles of about 36.2° and 33.6°, respectively. A model of how DHR38DBD binds to hsp27 in the presence of EcRDBD is presented. 相似文献
2.
I Grad A Niedziela-Majka M Kochman A Ozyhar 《European journal of biochemistry》2001,268(13):3751-3758
The steroid hormone, 20-hydroxyecdysone (20E), directs Drosophila metamorphosis via a heterodimeric receptor formed by two members of the nuclear hormone receptors superfamily, the product of the EcR (EcR) and of the ultraspiracle (Usp) genes. Our previous study [Niedziela-Majka, A., Kochman, M., Ozyhar, A. (2000) Eur. J. Biochem. 267, 507-519] on EcR and Usp DNA-binding domains (EcRDBD and UspDBD, respectively) suggested that UspDBD may act as a specific anchor that preferentially binds the 5' half-site of the pseudo-palindromic response element from the hsp27 gene promoter and thus locates the heterocomplex in the defined orientation. Here, we analyzed in detail the determinants of the UspDBD interaction with the hsp27 element. The roles of individual amino acids in the putative DNA recognition alpha helix and the roles of the base pairs of the UspDBD target sequence have been probed by site-directed mutagenesis. The results show how the hsp27 element specifies UspDBD binding and thus the polar assembly of the UspDBD/EcRDBD heterocomplex. It is suggested how possible nucleotide deviations within the 5' half-site of the element may be used for the fine-tuning of the 20E-response element specificity and consequently the physiological response. 相似文献
3.
The functional 20-hydroxyecdysone (20E) receptor is a heterodimer of two members of the nuclear hormone receptors superfamily; the product of the EcR (EcR) and of the ultraspiracle (Usp) genes. As most of the natural 20E-response elements are highly degenerated palindromes, we were interested in determining whether or not such asymmetric elements could dictate the defined orientation of the Usp/EcR complex. We have investigated interaction of EcR and Usp DNA-binding domains (EcRDBD and UspDBD, respectively) with the palindromic response element from the hsp27 gene promoter (hsp27pal). The hsp27pal half-sites contribute differently to the binding of the heterodimer components; the 5' half-site exhibits higher affinity for both DBDs than the 3' half-site. This observation, along with data demonstrating that UspDBD exhibits approximate fourfold higher affinity to the 5' half-site than EcRDBD, suggest that UspDBD locates the EcRDBD/UspDBD heterocomplex in the defined orientation (5'-UspDBD-EcRDBD-3') on the hsp27pal sequence. The binding polarity onto hsp27pal is accompanied by different contribution of the UspDBD and EcRDBD C-terminal sequences to the DNA-binding and heterocomplex formation. This is supported by finding that deletion of the C-terminal of EcRDBD region corresponding to the putative A-helix severely decreased binding of the EcRDBD to the hsp27pal. In contrast, UspDBD in which corresponding residues were deleted exhibited the same hsp27pal binding pattern as the wild type UspDBD. Additional truncation comprising the putative T-box, resulted in a reduced binding of the mutated UspDBD. This truncation however, still allowed effective EcRDBD/UspDBD heterodimer formation. Finally we demonstrated that perfect palindromes, composed of two hsp27pal 5' half-sites (or of the related sequence) contain all of the structural information necessary for the anisotropic UspDBD/EcRDBD heterocomplex formation. However, the perfect palindromes bind isolated homomeric DBDs as well as their heterocomplex with higher affinity than imperfect hsp27pal. This is the first report indicating that natural 20E response elements, which with one exception are degenerated palindromes, may act as functionally asymmetric elements in a manner similar to the action of direct repeats in vertebrates. 相似文献
4.
Markus Lezzi Thomas Bergman Vincent C Henrich Martin V?gtli Christina Fr?mel Marco Grebe Sabina Przibilla Margarethe Spindler-Barth 《European journal of biochemistry》2002,269(13):3237-3245
The insect ecdysteroid receptor consists of a heterodimer between EcR and the RXR-orthologue, USP. We addressed the question of whether this heterodimer, like all other RXR heterodimers, may be formed in the absence of ligand and whether ligand promotes dimerization. We found that C-terminal protein fragments that comprised the ligand binding, but not the DNA binding domain of EcR and USP and which were equipped with the activation or DNA binding region of GAL4, respectively, exhibit a weak ability to interact spontaneously with each other. Moreover, the heterodimer formation is greatly enhanced upon administration of active ecdysteroids in a dose-dependent manner. This was shown in vivo by a yeast two-hybrid system and in vitro by a modified electromobility shift assay. Furthermore, the EcR fragment expressed in yeast was functional and bound radioactively labelled ecdysteroid specifically. Ligand binding was greatly enhanced by the presence of a USP ligand binding domain. Therefore, ecdysteroids are capable of inducing heterodimer formation between EcR and USP, even when the binding of these receptor proteins to cognate DNA response elements does not occur. This capability may be a regulated aspect of ecdysteroid action during insect development. 相似文献
5.
Assembly of a glucocorticoid receptor complex prior to DNA binding enhances its specific interaction with a glucocorticoid response element 总被引:2,自引:0,他引:2
W Cairns C Cairns I Pongratz L Poellinger S Okret 《The Journal of biological chemistry》1991,266(17):11221-11226
Gel retardation analysis with full- and half-palindromic sequences using partially purified glucocorticoid receptor (GR) resulted in GR-glucocorticoid response element (GRE) species of identical mobilities, suggesting that formation of the dimeric GR protein complex is not catalyzed by DNA binding. These results are in contrast to the behavior of the isolated DNA binding domain of the glucocorticoid receptor where dimerization occurred on the GRE. Density gradient centrifugation of cytosolic GR resulted in two forms, a 4 S peak characteristic of the monomeric GR and a fraction which sediments at 6 S which is consistent with the observed size of the dimeric GR. These two forms were found to differ in their ability to bind to specific DNA sequences with the 6 S species having a higher affinity for a GRE. Taken together our results are consistent with a two-step model for hormone-induced transformation of GR: dissociation of the multimeric untransformed complex and dimerization of the GR to yield a high affinity DNA binding species. 相似文献
6.
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8.
J A Hengst-Zhang S A Weitzman 《Biochemical and biophysical research communications》1992,186(3):1515-1521
We have identified DNA binding proteins which interact with a sequence found in an intron of the tyrosine kinase coding portion of the murine c-abl gene. Several specific DNA: protein complexes were observed. Those complexes of approximate molecular weights 64 and 66kDa were detected when an Msp I site (CCGG) within the sequence was unmethylated, but were not observed when that site was methylated. Insertion of the intron sequence 5' to the rat somatic cytochrome C promoter and chloramphenicol acetyl transferase (CAT) sequences resulted in at least four-fold stimulation of CAT activity. These data suggest a potential role for the intron sequence in the regulation of gene expression. 相似文献
9.
Betanska K Nieva C Spindler-Barth M Spindler KD 《Archives of insect biochemistry and physiology》2007,65(3):134-142
The small G protein Ran, which is important for nucleocytoplasmic shuttling of proteins is present, but does not interact with EcR, Usp, and EcR/Usp. As shown by oligomycin treatment, EcR, Usp, and EcR/Usp import is energy dependent. Export of EcR and EcR/Usp is mediated by exportin-1 (CRM-1) as shown by the inhibiting effect of leptomycin B (LMB). Usp remains in the nucleus for more than 24 h. Nuclear retainment of EcR and Usp is energy dependent as shown by treatment with oligomycin. No export signal could be identified for Usp. The data confirm that EcR and Usp can enter the nucleus independently and that intracellular localization is regulated individually for each receptor. It is also demonstrated that the export signal of EcR is inaccessible after heterodimerization with Usp. 相似文献
10.
Replication protein A interactions with DNA: differential binding of the core domains and analysis of the DNA interaction surface 总被引:5,自引:0,他引:5
Human replication protein A (RPA) is a heterotrimeric (70, 32, and 14 kDa subunits), eukaryotic single-stranded DNA (ssDNA) binding protein required for DNA recombination, repair, and replication. The three subunits of human RPA are composed of six conserved DNA binding domains (DBDs). Deletion and mutational studies have identified a high-affinity DNA binding core in the central region of the 70 kDa subunit, composed of DBDs A and B. To define the roles of each DBD in DNA binding, monomeric and tandem DBD A and B domain chimeras were created and characterized. Individually, DBDs A and B have a very low intrinsic affinity for ssDNA. In contrast, tandem DBDs (AA, AB, BA, and BB) bind ssDNA with moderate to high affinity. The AA chimera had a much higher affinity for ssDNA than did the other tandem DBDs, demonstrating that DBD A has a higher intrinsic affinity for ssDNA than DBD B. The RPA-DNA interface is similar in both DBD A and DBD B. Mutational analysis was carried out to probe the relative contributions of the two domains to DNA binding. Mutation of polar residues in either core DBD resulted in a significant decrease in the affinity of the RPA complex for ssDNA. RPA complexes with pairs of mutated polar residues had lower affinities than those with single mutations. The decrease in affinity observed when polar mutations were combined suggests that multiple polar interactions contribute to the affinity of the RPA core for DNA. These results indicate that RPA-ssDNA interactions are the result of binding of multiple nonequivalent domains. Our data are consistent with a sequential binding model for RPA, in which DBD A is responsible for positioning and initial binding of the RPA complex while DBD A together with DBD B direct stable, high-affinity binding to ssDNA. 相似文献
11.
Carolyn M. Klinge Colleen L. Brolly Robert A. Bambara Russell Hilf 《The Journal of steroid biochemistry and molecular biology》1997,63(4-6):283-301
Bovine estrogen receptor (ER) was purified to near homogeneity by estrogen response element (ERE) affinity chromatography, and its ERE binding ability was measured in vitro. Highly purified ER bound EREs with reduced affinity compared to partially purified ER. Partially purified ER contained hsp70, but highly purified ER did not. We examined whether addition of purified recombinant human hsp70 or purified bovine hsp70 would restore the higher ERE binding affinity, stoichiometry, and ligand retention detected with partially purified receptor and how hsp70 affected the rate of ER-ERE association and dissociation. ER-ERE binding was not affected by antibodies to either constitutive or induced forms of hsp70, regardless of ER purity. Addition of purified hsp70, with or without ATP and Mg2+, did not affect the association or dissociation rates of highly purified liganded ER binding to ERE. hsp70 Did not alter the total amount of ER-ERE complex formed. Similarly, hsp70 did not affect the rate of [3H]estradiol (E2) or [3H]4-hydroxytamoxifen (4-OHT) ligand dissociation from ER in the presence or absence of EREs. These data contrast with a report showing that maximal ERE binding by highly purified recombinant human ER required hsp70. We conclude that ER, purified from a physiological source, i.e., calf uterus, does not require hsp70 for maximal ER-ERE binding in vitro. Additionally, once ER is activated and bound by ligand, the receptor assumes its proper tertiary structure, and hsp70 does not impact ER ligand binding domain conformation. 相似文献
12.
Estrogen regulation of the rat prolactin gene requires sequences within the DNase I hypersensitive site II (HSII). We have used overexpressed mouse estrogen receptor alpha (ERalpha) protein to study interactions of ERalpha with an imperfect estrogen response element (ERE) and four ERE half-site sequences from HSII. We confirmed that ERalpha has higher affinity for ERE half-sites than for the imperfect ERE. As expected, the imperfect ERE formed a complex with ERalpha similar to that between mERalpha and a consensus ERE in gel shift assays. The ERalpha complex with half-sites, however, had faster mobility on a 4% polyacrylamide gel than the ERalpha complex with a consensus ERE, indicating that the complexes had different compositions. Ferguson analysis revealed that the ERalpha/half-site complex had a larger molecular weight and higher negative charge than the ERalpha/consensus ERE complex. Similar results were observed with purified human ERalpha, showing that the ERalpha/half-site complex contained only ERalpha and oligonucleotides. These results are best explained by a model in which a dimer of ERalpha is bound to two half-site oligonucleotides. We propose that two ERalpha dimers may interact with the four ERE half-sites in HSII to influence estrogen regulation of this gene. 相似文献
13.
The crystal structure of a fucose-binding lectin from the bacteria Pseudomonas aeruginosa in complex with α-L-fucose has been recently determined. It is a tetramer; each monomer displays a nine-stranded, antiparallel,
β-sandwiched arrangement and contains two calcium ions that mediate the binding of fucose in a recognition mode unique among
protein-carbohydrate interactions. In search of this type of unique interactions in other newly discovered protein sequences,
we have used molecular modeling techniques to predict and analyze the 3-D structures of some proteins, which exhibited reasonable
degree of homology with the amino acid sequence of the bacterial protein. A BLAST search with the sequence of Pseudomonas aeruginosa as query in the non-redundant sequence database identified four proteins from different species, three organisms from bacteria
and one from archaea. We have modeled the structures of these proteins as well as those of the complexes with carbohydrates
and studied the nature of physicochemical forces involved in the complex formation both in presence and absence of calcium.
The calcium-binding loops have been found to be highly conserved both in terms of primary and tertiary structures in these
proteins, although a less acidic character is observed in Photorhabdus lectin due to the absence of two aspartic acid residues on the calcium-binding loop which also resulted in lower binding
affinity. All these structures exhibited highly negative electrostatic environment in the vicinity of the calcium-binding
loops which was essential for neutralizing the positive charges of two closely situated Ca+2 ions. The comparison of the binding affinities of some monosaccharides other than fucose, e.g. mannose and fructose, showed
higher binding energies confirming the fucose specificity of these proteins. 相似文献
14.
Epand RM 《Progress in lipid research》2006,45(4):279-294
Cholesterol is not uniformly distributed in biological membranes. One of the factors influencing the formation of cholesterol-rich domains in membranes is the unequal lateral distribution of proteins in membranes. Certain proteins are found in cholesterol-rich domains. In some of these cases, it is as a consequence of the proteins interacting directly with cholesterol. There are several structural features of a protein that result in the protein preferentially associating with cholesterol-rich domains. One of the best documented of these is certain types of lipidations. In addition, however, there are segments of a protein that can preferentially sequester cholesterol. We discuss two examples of these cholesterol-recognition elements: the cholesterol recognition/interaction amino acid consensus (CRAC) domain and the sterol-sensing domain (SSD). The requirements for a CRAC motif are quite flexible and predict that a large number of sequences could recognize cholesterol. There are, however, certain proteins that are known to interact with cholesterol-rich domains of cell membranes that have CRAC motifs, and synthetic peptides corresponding to these segments also promote the formation of cholesterol-rich domains. Modeling studies have provided a rationale for certain requirements of the CRAC motif. The SSD is a larger protein segment comprising five transmembrane domains. The amino acid sequence YIYF is found in several SSD and in certain other proteins for which there is evidence that they interact with cholesterol-rich domains. The CRAC sequences as well as YIYF are generally found adjacent to a transmembrane helical segment. These regions appear to have a strong influence of the localization of certain proteins into domains in biological membranes. In addition to the SSD, there is also a domain found in soluble proteins, the START domain, that binds lipids. Certain proteins with START domains specifically bind cholesterol and are believed to function in intracellular cholesterol transport. One of these proteins is StAR-D1, that also has a mitochondrial targeting sequence and plays an important role in delivering cholesterol to the mitochondria of steroidogenic cells. 相似文献
15.
Moaddel R Price GB Juteau JM Leffak M Wainer IW 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2005,820(2):197-203
The DNA unwinding element binding protein (DUE-B) plays a key role in DNA replication. The DUE-B protein has been immobilized on a liquid chromatography support and the resulting immobilized protein column was used for the on-line screening of a series of steroids. The DUE-B protein was expressed with an added C-terminal sequence of six adjacent histidine residues, a His6-tag and immobilized on a chiral ligand exchange support, the CLC-L column, using Ni2+ as the coordinating metal ion. The chromatographic retentions of 12 steroids were determined on the DUE-B/CLC-L column. The magnitudes of the steroid-immobilized DUE-B interactions, reflected by the observed retention times, correlated to the effect of the steroids in the cell-free replication system, i.e. the longer the retention, the greater the increase in DNA replication. The coefficient of determination for the %DNA activities linear relation to retention time was 0.9694. The data suggest that the DUE-B/CLC-L phase can be used for on-line pharmacological studies. The results also indicated that His-tagged proteins can be directly immobilized on the CLC-L stationary phase and the resulting columns used as rapid screens for the isolation and identification of small molecule or protein ligands from complex biological or chemical mixtures. 相似文献
16.
The amino terminus of Tax is required for interaction with the cyclic AMP response element binding protein. 总被引:4,自引:5,他引:4
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Tax of human T-cell leukemia virus type 1 was analyzed for interaction with the cyclic AMP response element binding protein (CREB) in vitro with and without Tax response element DNA. Mutations in the carboxy terminus of Tax (L296G and L320G) did not affect binding to CREB and led to supershifts. In contrast, mutants with changes in the amino-terminal cysteine-rich region lost the ability to bind to CREB. The S10A mutant protein bound moderately. Thus, the amino terminus of Tax is essential for Tax-CREB interaction. 相似文献
17.
Palmer MJ Warren JT Jin X Guo X Gilbert LI 《Insect biochemistry and molecular biology》2002,32(4):465-476
Total body ecdysteroid titers were determined at specific stages during the larval and nymphal life of Amblyomma americanum (L.). One ecdysteroid peak was observed following the completion of larval apolysis. However, two distinct ecdysteroid peaks occurred at a comparable stage in the nymphal molting cycle. The first occurred following apolysis and the second peak occurred at about the time of ecdysis. When whole body profiles of EcR and RXR mRNAs were examined during the molting cycle using RT-PCR, the expression of both AamEcR and AamRXR mRNAs was shown to be correlated with the ecdysteroid titer. Using an electrophoretic gel mobility shift assay, it was demonstrated that AamEcR*AamRXR1, but not AamEcR*AamRXR2, exhibits broad DNA binding specificity, forming complexes with a variety of synthetic direct repeat and palindromic nuclear response elements with the half-site consensus AGGTCA. These data suggest that functional differences may exist between the AamRXR1 and AamRXR2 proteins. 相似文献
18.
1H NMR studies of DNA recognition by the glucocorticoid receptor: complex of the DNA binding domain with a half-site response element. 总被引:1,自引:0,他引:1
M L Remerowski E Kellenbach R Boelens G A van der Marel J H van Boom B A Maler K R Yamamoto R Kaptein 《Biochemistry》1991,30(50):11620-11624
The complex of the rat glucocorticoid receptor (GR) DNA binding domain (DBD) and half-site sequence of the consensus glucocorticoid response element (GRE) has been studied by two-dimensional 1H NMR spectroscopy. The DNA fragment is a 10 base-pair oligonucleotide, 5'd(GCTGTTCTGC)3'.5'd-(GCAGAACAGC)3', containing the stronger binding GRE half-site hexamer, with GC base pairs at each end. The 93-residue GR-DBD contains an 86-residue segment corresponding to residues 440-525 of the rat GR. Eleven NOE cross peaks between the protein and DNA have been identified, and changes in the chemical shift of the DNA protons upon complex formation have been analyzed. Using these protein-DNA contact points, it can be concluded that (i) the "recognition helix" formed by residues C460-E469 lies in the major groove of the DNA; (ii) the GR-DBD is oriented on the GRE half-site such that residues A477-D481, forming the so-called D-loop, are available for protein-protein interaction in the GR-DBD dimer on the intact consensus GRE; and (iii) the 5-methyl of the second thymine in the half-site and valine 462 interact, confirming indirect evidence [Truss et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7180-7184; Mader et al. (1989) Nature 338, 271-274] that both play an important role in GR-DBD DNA binding. These findings are consistent with the model proposed by H?rd et al. [(1990) Science 249, 157-160] and the X-ray crystallographic complex structure determined by Luisi et al. [(1991) Nature 352, 497-505]. 相似文献
19.
Vishnivetskiy SA Gimenez LE Francis DJ Hanson SM Hubbell WL Klug CS Gurevich VV 《The Journal of biological chemistry》2011,286(27):24288-24299
Arrestins bind active phosphorylated forms of G protein-coupled receptors, terminating G protein activation, orchestrating receptor trafficking, and redirecting signaling to alternative pathways. Visual arrestin-1 preferentially binds rhodopsin, whereas the two non-visual arrestins interact with hundreds of G protein-coupled receptor subtypes. Here we show that an extensive surface on the concave side of both arrestin-2 domains is involved in receptor binding. We also identified a small number of residues on the receptor binding surface of the N- and C-domains that largely determine the receptor specificity of arrestins. We show that alanine substitution of these residues blocks the binding of arrestin-1 to rhodopsin in vitro and of arrestin-2 and -3 to β2-adrenergic, M2 muscarinic cholinergic, and D2 dopamine receptors in intact cells, suggesting that these elements critically contribute to the energy of the interaction. Thus, in contrast to arrestin-1, where direct phosphate binding is crucial, the interaction of non-visual arrestins with their cognate receptors depends to a lesser extent on phosphate binding and more on the binding to non-phosphorylated receptor elements. 相似文献