Hydrophobic cluster analysis (HCA) of the hormone-binding domain of receptor proteins

A new technique of protein sequence analysis, namely, Hydrophobic Cluster Analysis (HCA), has been used to align and compare the sequences of proteins belonging to the receptor superfamily (steroid, thyroid hormone and retinoic acid receptors) and serpin superfamily (corticosteroid binding globulin (CBG) and alpha 1-antitrypsin (alpha 1-AT]. By matching up clusters of hydrophobic amino-acids that oftenmost correspond to identifiable secondary structures (alpha-helices, beta-strands etc.), it has been possible to deduce the following information on the secondary structures of these proteins: CBG is structurally related to alpha 1-AT (HCA score greater than 80%), the structures of the hormone-binding domains of the steroid receptors that bind 3-keto-delta 4-steroids are closely interrelated (greater than 80%) but less closely related to that of the estrogen receptor (ER) (approximately 75%), vitamin D, retinoic acid and thyroid hormone receptors are structurally closely related (greater than or equal to 80%). Their secondary structures are, however, also related to that of the steroid receptors (approximately 70%), and a high degree of analogy exists between the structures of serpins and of the hormone-binding domains of members of the steroid superfamily (60-70%). HCA has clearly shown that a previous local sequence alignment of the estrogen receptor with other steroid receptors and cytochromes P450 has to be reconsidered. The published consensus steroid binding sequence previously identified in cytochromes is in fact 80 amino-acids upstream from its previously defined position. Other regions of contiguous sequence identity have also been identified which may be involved in the hydrophobic core of the protein or in steroid binding. Their positions have been indicated using the crystal structure of alpha 1-AT as a model.     

Molecular cloning of a steroid-regulated 108K heat shock protein gene from hen oviduct.

The natural gene for a steroid inducible 108K heat shock protein has been isolated from a lambda genomic library prepared from hen oviduct tissue. Genomic DNA blots indicate that it exists as a single copy gene in the chick oviduct haploid genome. The 9.9 kilobase gene codes for a messenger RNA of 2733bp (21) and is split into 18 exons as established by sequence comparison of cDNA and genomic clones. The 3' end of the gene contains a repetitive element which shares homology with the CR1 family of repeats. The first exon contains both the untranslated leader and coding regions of the gene. The promoter region is rich in G + C residues (70%) and the dinucleotide CG. This 5' flanking segment contains bases similar both in sequence and location to the Goldberg-Hogness TATA homology and consensus sequence CCAAT. A consensus sequence located upstream of steroid hormone responsive chicken genes is found at -267 and on a reverse orientation at -593. The structure of this gene is of interest since the presence of introns in heat shock genes is rare in any species examined to date. Furthermore, this gene lacks the previously described heat shock promoter consensus sequence (C-GAA-TTC-G) present in other species.     

In vitro studies on the role of the peripheral-type benzodiazepine receptor in steroidogenesis

In vitro studies using isolated cells, mitochondria and submitochondrial fractions demonstrated that in steroid synthesizing cells, the peripheral-type benzodiazepine receptor (PBR) is an outer mitochondrial membrane protein, preferentially located in the outer/inner membrane contact sites, involved in the regulation of cholesterol transport from the outer to the inner mitochondrial membrane, the rate-determining step in steroid biosynthesis. Mitochondrial PBR ligand binding characteristics and topography are sensitive to hormone treatment suggesting a role of PBR in the regulation of hormone-mediated steroidogenesis. Targeted disruption of the PBR gene in Leydig cells in vitro resulted in the arrest of cholesterol transport into mitochondria and steroid formation; transfection of the mutant cells with a PBR cDNA rescued steroidogenesis demonstrating an obligatory role for PBR in cholesterol transport. Molecular modeling of PBR suggested that it might function as a channel for cholesterol. This hypothesis was tested in a bacterial system devoid of PBR and cholesterol. Cholesterol uptake and transport by these cells was induced upon PBR expression. Amino acid deletion followed by site-directed mutagenesis studies and expression of mutant PBRs demonstrated the presence in the cytoplasmic carboxy-terminus of the receptor of a cholesterol recognition/interaction amino acid consensus sequence. This amino acid sequence may help for recruiting the cholesterol coming from intracellular sites to the mitochondria.     

Inhibition of protein interactions with the beta 2 sliding clamp of Escherichia coli DNA polymerase III by peptides from beta 2-binding proteins

The sliding clamp of the Escherichia coli replisome is now understood to interact with many proteins involved in DNA synthesis and repair. A universal interaction motif is proposed to be one mechanism by which those proteins bind the E. coli sliding clamp, a homodimer of the beta subunit, at a single site on the dimer. The numerous beta(2)-binding proteins have various versions of the consensus interaction motif, including a related hexameric sequence. To determine if the variants of the motif could contribute to the competition of the beta-binding proteins for the beta(2) site, synthetic peptides derived from the putative beta(2)-binding motifs were assessed for their abilities to inhibit protein-beta(2) interactions, to bind directly to beta(2), and to inhibit DNA synthesis in vitro. A hierarchy emerged, which was consistent with sequence similarity to the pentameric consensus motif, QL(S/D)LF, and peptides containing proposed hexameric motifs were shown to have activities comparable to those containing the consensus sequence. The hierarchy of peptide binding may be indicative of a competitive hierarchy for the binding of proteins to beta(2) in various stages or circumstances of DNA replication and repair.     

Protein-DNA interactions at recognition sites for the dioxin-Ah receptor complex

Gel retardation analyses reveal a cluster of six binding sites for the liganded Ah receptor within a 700-base pair DNA domain upstream of the mouse CYP1A1 gene. The nucleotide sequences of the binding sites define a consensus recognition motif for the liganded receptor. The consensus motif is not symmetric. Alteration of the consensus motif produces a decrease in the receptor-DNA interaction. The ligand receptor binds as a monomer to its recognition motif and preferentially binds to double-stranded DNA. These observations reveal apparent differences between 2,3,7,8-tetrachlorodibenzo-p-dioxin and steroid hormones in their respective mechanisms of action.     

C-terminal sequences outside the tetratricopeptide repeat domain of FKBP51 and FKBP52 cause differential binding to Hsp90

Hsp90 assembles with steroid receptors and other client proteins in association with one or more Hsp90-binding cochaperones, some of which contain a common tetratricopeptide repeat (TPR) domain. Included in the TPR cochaperones are the Hsp70-Hsp90-organizing protein Hop, the FK506-binding immunophilins FKBP52 and FKBP51, the cyclosporin A-binding immunophilin CyP40, and protein phosphatase PP5. The TPR domains from these proteins have similar x-ray crystallographic structures and target cochaperone binding to the MEEVD sequence that terminates Hsp90. However, despite these similarities, the TPR cochaperones have distinctive properties for binding Hsp90 and assembling with Hsp90.steroid receptor complexes. To identify structural features that differentiate binding of FKBP51 and FKBP52 to Hsp90, we generated an assortment of truncation mutants and chimeras that were compared for coimmunoprecipitation with Hsp90. Although the core TPR domain (approximately amino acids 260-400) of FKBP51 and FKBP52 is required for Hsp90 binding, the C-terminal 60 amino acids (approximately 400-end) also influence Hsp90 binding. More specifically, we find that amino acids 400-420 play a critical role for Hsp90 binding by either FKBP. Within this 20-amino acid region, we have identified a consensus sequence motif that is also present in some other TPR cochaperones. Additionally, the final 30 amino acids of FKBP51 enhance binding to Hsp90, whereas the corresponding region of FKBP52 moderates binding to Hsp90. Taking into account the x-ray crystal structure for FKBP51, we conclude that the C-terminal regions of FKBP51 and FKBP52 outside the core TPR domains are likely to assume alternative conformations that significantly impact Hsp90 binding.     

Defining the sequence recognized with BmFTZ-F1, a sequence specific DNA binding factor in the silkworm, Bombyx mori, as revealed by direct sequencing of bound oligonucleotides and gel mobility shift competition analysis.

BmFTZ-F1 is a Bombyx mori homologue of FTZ-F1, a positive regulator of the fushi tarazu gene of Drosophila melanogaster. In order to determine the sequence recognized with this factor, we made three sets of oligonucleotide mixture which contain 4 possible nucleotides at different positions within the previously proposed 12-bp binding consensus sequence. Oligonucleotides which bound to purified BmFTZ-F1 were separated by a gel mobility shift procedure and a binding sequence was determined by direct sequencing through Maxam-Gilbert method. By this analysis, 7 positions showed clear sequence preference and 5 positions showed weak or no sequence preference. The importance of each nucleotide at each position was confirmed by a gel mobility shift competition analysis and results were presented as a quantitative difference in the binding affinity. From these analyses, we conclude that the best binding sequence of BmFTZ-F1 is 5'-PyCAAGGPyCPu-3'. This method may be useful for the determination of a binding sequence of other sequence specific DNA binding factor.     

The PurR binding site in the glyA prometer region of Escherichia coli

Site-directed mutagenesis was used to change the PurR binding site in the control region of a glyA-lac gene fusion. Mutations that changed the PurR binding sequence away from the consensus sequence reduced PurR binding, which correlated with reduced purine-mediated repression. Mutations that changed the binding sequence toward the consensus sequence had no significant effect on either PurR binding or purine-mediated repression. Hypoxanthine and guanine, co-repressors for PurR-mediated regulation of the pur regulon, increased binding of PurR to glyA operator DNA.     

Specific and nonspecific interactions of integration host factor with oligo DNAs as revealed by circular dichroism spectroscopy and filter binding assay.

Binding specificity of integration host factor (IHF) to oligo DNAs has been studied by circular dichroism (CD) spectroscopy and filter binding experiment. CD difference spectra of IHF-DNA complexes demonstrated that a conformational change in DNA was induced by binding of IHF when DNA had a consensus sequence for the binding sites of IHF, but that such conformational change was not observed for consensus DNA 20 mer as well as nonconsensus DNA 45 mer. Dissociation constants for IHF-DNA complexes determined by filter binding assay showed that IHF has indeed stronger affinity to DNA with the consensus binding site than to nonconsensus DNA, but the difference in its affinity between consensus and nonconsensus DNAs was rather small, 3.4-fold. It was, therefore, concluded that the flanking regions of the consensus sequence are important for the specific binding of IHF and that its binding specificity is well characterized by the induced conformational change in DNA rather than by dissociation constants for IHF-DNA complexes.     

A-type ATP binding consensus sequences are critical for the catalytic activity of the calmodulin-sensitive adenylyl cyclase from Bacillus anthracis

Analysis of the predicted amino acid sequence of Bacillus anthracis adenylyl cyclase revealed sequences with homology to consensus sequences for A- and B-type ATP binding domains found in many ATP binding proteins. Based on the analysis of nucleotide binding proteins, a conserved basic amino acid residue in the A-type consensus sequence and a conserved acidic amino acid residue in the B-type consensus sequence have been implicated in the binding of ATP. The putative ATP binding sequences in the B. anthracis adenylyl cyclase possess analogous lysine residues at positions 346 and 353 within two A-type consensus sequences and a glutamate residue at position 436 within a B-type consensus sequence. The two A-type consensus sequences overlap each other and have the opposite orientation. To determine whether Lys-346, Lys-353, or Glu-436 of the B. anthracis adenylyl cyclase are crucial for enzyme activity, Lys-346 and Lys-353 were replaced with methionine and Glu-436 with glutamine by oligonucleotide-directed mutagenesis. Furthermore, Lys-346 was also replaced with arginine. The genes encoding the wild type and mutant adenylyl cyclases were placed under the control of the lac promoter for expression in Escherichia coli, and extracts were assayed for adenylyl cyclase activity. In all cases, a 90-kDa polypeptide corresponding to the catalytic subunit of the enzyme was detected in E. coli extracts by rabbit polyclonal antibodies raised against the purified B. anthracis adenylyl cyclase. The proteins with the Lys-346 to methionine or arginine mutations exhibited no adenylyl cyclase activity, indicating that Lys-346 in the A-type ATP binding consensus sequence plays a critical role for enzyme catalysis. Furthermore, the enzyme with the Lys-353 to methionine mutation was also inactive, suggesting that Lys-353 may also directly contribute to enzyme catalysis. In contrast, the protein with the Glu-436 to glutamine mutation retained 75% of enzyme activity, suggesting that Glu-436 in the B-type ATP binding consensus sequence may not be directly involved in enzyme catalysis. It is concluded that Lys-346 and Lys-353 in B. anthracis adenylyl cyclase may interact directly with ATP and contribute to the binding of the nucleotide to the enzyme.     

Corticosteroid binding globulin, testosterone-estradiol binding globulin, and androgen binding protein belong to protein families distinct from steroid receptors

The cDNA nucleotide sequences and the deduced amino acid sequences of human corticosteroid binding globulin (hCBG), human testosterone-estradiol binding globulin (hTeBG), and rat androgen binding protein (rABP) were determined. Studies of the steroid binding sites suggest they are toward the carboxy-terminus in hTeBG and rABP and more central in hCBG. hCBG has remarkable sequence homology with members of a superfamily whose functions have diverged; these include thyroxine-binding protein, serine protease inhibitors, egg white proteins, and angiotensinogen. hTeBG and rABP have a 68% amino acid sequence identity. Hybridization studies suggest that hTeBG is probably even more closely related, if not identical, to hABP. The carboxy-terminal sequences of hTeBG and rABP are also similar to that of protein S, a vitamin-K-dependent clotting factor. There were no nucleotide or amino acid sequence homologies between hCBG, hTeBG, or rABP and other steroid binding proteins such as steroid receptors, albumin, alpha-fetoprotein, and vitamin D binding protein. We conclude that the "extracellular steroid binding proteins" and steroid receptors do not appear to have descended from a common ancestor.     

Identification of low density lipoprotein receptor binding domains of human apolipoprotein B-100: a proposed consensus LDL receptor binding sequence of apoB-100

The human liver apoB-100 gene cloned in the lambda gt-11 expression vector expresses fusion proteins reacting with apoB antibodies. A fusion protein induced from a apoB-lambda gt-11 clone reacted with apoB-100 monoclonal antibodies known to block the binding of LDL to the LDL receptor. The fusion protein contains an amino acid sequence domain enriched in positively charged residues which is complementary to the negatively charged amino acids present in the consensus LDL receptor binding domain. This sequence of apoB-100 is proposed as a binding domain for the interaction with the LDL receptor. Comparison of derived amino acid sequences from the entire structure of apoB-100 molecule revealed several similar domains enriched in positively charged amino acids. A consensus sequence of the potential LDL binding domain was identified which contained positively charged amino acids at positions 1, 5 and 8 and a loop of 8-11 amino acids followed by two adjacent positively charged amino acids. These results are interpreted as indicating that there are several potential LDL receptor binding domains in apoB-100.