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Query: UNIPROT:P06889 (Mol)
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A series of human androgen receptor (AR) deletion mutants was constructed to study the relationship between various structural domains and their different functions in the AR protein. Immunoblots of wild type AR and AR mutants expressed in COS-1 cells, revealed a doublet appearance of all AR proteins. One exception was an AR mutant lacking amino acid residues 51-211 that migrated as a single protein band, possibly due to altered post-translational modification. The steroid binding domain was found to be encoded by approx. 250 amino acid residues in the C-terminal end. Deletions and truncations in this part of the receptor abolished hormone binding. The N-terminal domain was observed to be essential for transcriptional activation. AR mutants lacking large parts of this domain were transcriptionally inactive. Deletion of the hormone binding domain yielded a constitutively active AR protein, indicating that in the absence of hormone this domain displays an inhibitory function. In the absence of ligand the wild type AR expressed in COS-1 cells was distributed over nucleus and cytoplasm. The addition of hormone directed all androgen receptors to the nucleus. In contrast, an AR mutant lacking part of the DNA binding domain and part of the hinge region, was almost exclusively cytoplasmic in the absence of hormone. This mutant lacks a conserved region, homologous to the SV40 large T- and nucleoplasmin nuclear localization signal. Hormone induced transfer of this AR mutant to the nucleus, indicating the presence of a second, hormone dependent nuclear targeting mechanism.
J Steroid Biochem Mol Biol 1992 Mar
PMID:Functional domains of the human androgen receptor. 156 40

The crystal structure of the complex between bovine alpha-chymotrypsin and the leech (Hirudo medicinalis) protein proteinase inhibitor eglin c has been refined at 2.0 A resolution to a crystallographic R-factor of 0.167. The structure of the complex includes 2290 protein and 143 solvent atoms. Eglin c is bound to the cognate enzyme through interactions involving 11 residues of the inhibitor (sites P5-P4' in the reactive site loop, P10' and P23') and 17 residues from chymotrypsin. Binding of eglin c to the enzyme causes a contained hinge-bending movement around residues P4 and P4' of the inhibitor. The tertiary structure of chymotrypsin is little affected, with the exception of the 10-13 region, where an ordered structure for the polypeptide chain is observed. The overall binding mode is consistent with those found in other serine proteinase-protein-inhibitor complexes, including those from different inhibition families. Contained, but significant differences are observed in the establishment of intramolecular hydrogen bonds and polar interactions stabilizing the structure of the intact inhibitor, if the structure of eglin c in its complex with chymotrypsin is compared with that of other eglin c-serine proteinase complexes.
J Mol Biol 1992 May 05
PMID:Crystal and molecular structure of the bovine alpha-chymotrypsin-eglin c complex at 2.0 A resolution. 158 84

The X-ray structure of the periplasmic ribose receptor (binding protein) of Escherichia coli (RBP) was solved at 3 A resolution by the method of multiple isomorphous replacement. Alternating cycles of refitting and refinement have resulted in a model structure with an R-factor of 18.7% for 27,526 reflections from 7.5 to 1.7 A resolution (96% of the data). The model contains 2228 non-hydrogen atoms, including all 271 residues of the amino acid sequence, 220 solvent atoms and beta-D-ribose. The protein consists of two highly similar structural domains, each of which is composed of a core of parallel beta-sheet flanked on both sides by alpha-helices. The two domains are related to each other by an almost perfect 2-fold axis of rotation, with the C termini of the beta-strands of each sheet pointing toward the center of the molecule. Three short stretches of amino acid chain (from symmetrically related portions of the protein) link these two domains, and presumably act as a hinge to allow relative movement of the domains in functionally important conformational changes. Two water molecules are also an intrinsic part of the hinge, allowing crucial flexibility in the structure. The ligand beta-D-ribose (in the pyranose form) is bound between the domains, held by interactions with side-chains of the interior loops. The binding site is precisely tailored, with a combination of hydrogen bonding, hydrophobic and steric effects giving rise to tight binding (0.1 microM for ribose) and high specificity. Four out of seven binding-site residues are charged (2 each of aspartate and arginine) and contribute two hydrogen bonds each. The remaining hydrogen bonds are contributed by asparagine and glutamine residues. Three phenylalanine residues supply the hydrophobic component, packing against both faces of the sugar molecule. The arrangement of these hydrogen bonding and hydrophobic residues results in an enclosed binding site with the exact shape of the allowed sugar molecules; in the process of binding, the ligand loses all of its surface-accessible area. The sites of two mutations that affect the rate of folding of the ribose receptor are shown to be located near small cavities in the wild-type protein. The cavities thus allow the incorporation of the larger residues in the mutant proteins. Since these alterations would seriously affect the ability of the protein to build the first portion of the hydrophobic core in the first domain, it is proposed that this process is the rate-limiting step in folding of the ribose receptor.
J Mol Biol 1992 May 05
PMID:1.7 A X-ray structure of the periplasmic ribose receptor from Escherichia coli. 158 88

The dynamic structure of a protein, human lysozyme, is determined by the normal mode refinement of X-ray crystal structure. This method uses the normal modes of both internal and external motions to distinguish the real internal dynamics from the external terms such as lattice disorder, and gives an anisotropic and concerted picture of atomic fluctuations. The refinement is carried out with diffraction data of 5.0 to 1.8 A resolution, which are collected on an imaging plate. The results of the refinement show: (1) Debye-Waller factor consists of two parts, highly anisotropic internal fluctuations and almost isotropic external terms. The former is smaller than the latter by a factor of 0.72 in the scale of B-factor. Therefore, the internal dynamics cannot be recognized directly from the apparent electron density distribution. (2) The internal fluctuations show basically similar features as those predicted by the normal mode analysis, with almost the same amplitude and a similar level of anisotropy. (3) Correlations of fluctuations are detected between two lobes forming the active site cleft, which move simultaneously in opposite directions. This corresponds to the hinge-bending motion of lysozyme.
J Mol Biol 1992 May 20
PMID:Normal mode refinement: crystallographic refinement of protein dynamic structure. II. Application to human lysozyme. 159 31

The conformation of Escherichia coli 5 S rRNA was investigated using chemical and enzymatic probes. The four bases were monitored at one of their Watson-Crick positions with dimethylsulfate (at C(N-3) and A(N-1], with a carbodiimide derivative (at G(N-1) and U(N-3] and with kethoxal (at G(N-1, N-2]. Position N-7 of purine was probed with diethylpyrocarbonate (at A(N-7] and dimethylsulfate (at G(N-7]. Double-stranded or stacked regions were tested with RNase V1 and unpaired guanine residues with RNase T1. We also used lead(II) that has a preferential affinity for interhelical and loop regions and a high sensitivity for flexible regions. Particular care was taken to use uniform conditions of salt, magnesium, pH and temperature for the different enzymatic chemical probes. Derived from these experimental data, a three dimensional model of the 5 S rRNA was built using computer modeling which integrates stereochemical constraints and phylogenetic data. The three domains of 5 S rRNA secondary structure fold into a Y-shaped structure that does not accommodate long-range tertiary interactions between domains. The three domains have distinct structural and dynamic features as revealed by the chemical reactivity and the lead(II)-induced hydrolysis: domain 2 (loop B/helix III/loop C) displays a rather weak structure and possesses dynamic properties while domain 3 (helix V/region E/helix IV/loop D) adopts a highly structured and overall helical conformation. Conserved nucleotides are not crucial for the tertiary folding but maintain an intrinsic structure in the loop regions, especially via non-canonical pairing (A.G, G.U, G.G, A.C, C.C), which can close the loops in a highly specific fashion. In particular, nucleotides in the large external loop C fold into an organized conformation leading to the formation of a five-membered loop motif. Finally, nucleotides at the hinge region of the Y-shape are involved in a precise array of hydrogen bonds based on a triple interaction between U14, G69 and G107 stabilizing the quasi-colinearity of helices II and V. The proposed tertiary model is consistent with the localization of the ribosomal protein binding sites and possesses strong analogy with the model proposed for Xenopus laevis 5 S rRNA, indicating that the Y-shape model can be generalized to all 5 S rRNAs.
J Mol Biol 1991 Sep 05
PMID:Three-dimensional model of Escherichia coli ribosomal 5 S RNA as deduced from structure probing in solution and computer modeling. 171 95

The plasmid pBRD026, which directs expression of the B subunit of the Escherichia coli heat-labile toxin (LTB), was modified so that DNA encoding epitopes could be inserted at the 3' end of the gene. An oligonucleotide linker containing restriction sites for BglII and SpeI was inserted at the SpeI site at the 3' end of the LTB gene to form plasmid pFV1. This linker also encodes the amino acid sequence Gly-Pro-Gly-Pro which we propose acts as a 'hinge' between the LTB and the foreign epitope. Oligonucleotides specifying an epitope from the Bordetella pertussis P.69 outer membrane protein were cloned into pFV1 to form pFV169. The resultant fusion protein (LTB69) was partially purified from the periplasm of E. coli strains in a soluble pentameric form which could bind GM1 gangliosides. Mice immunized intranasally with purified LTB69 produced antibodies against both LTB and the P.69 protein. In addition, ELISPOT assays demonstrated the presence of LTB-specific and P.69-specific antibody-secreting cells in the lungs of immunized mice.
Mol Microbiol 1991 Jun
PMID:Intranasal immunization using the B subunit of the Escherichia coli heat-labile toxin fused to an epitope of the Bordetella pertussis P.69 antigen. 172 57

The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for yeast cell growth and division. To identify additional components of the pathway in which PKC1 functions, we isolated extragenic suppressors of a pkc1 deletion mutant. All of the suppressor mutations were dominant for suppressor function and defined a single locus, which was designated BCK1 (for bypass of C kinase). A molecular clone of one suppressor allele, BCK1-20, was isolated on a centromere-containing plasmid through its ability to rescue a conditional pkc1 mutant. The BCK1 gene possesses a 4.4-kb uninterrupted open reading frame predicted to encode a 163-kDa protein kinase. The BCK1 gene product is not closely related to any known protein kinase, sharing only 45% amino acid identity with its closest known relative (the STE11-encoded protein kinase) through a region restricted to its putative C-terminal catalytic domain. Deletion of BCK1 resulted in a temperature-sensitive cell lysis defect, which was suppressed by osmotic stabilizing agents. Because pkc1 mutants also display a cell lysis defect, we suggest that PKC1 and BCK1 may normally function within the same pathway. Suppressor alleles of BCK1 differed from the wild-type gene in a region surrounding a potential PKC phosphorylation site immediately upstream of the predicted catalytic domain. This region may serve as a hinge between domains whose interaction is regulated by PKC1.
Mol Cell Biol 1992 Jan
PMID:Dominant mutations in a gene encoding a putative protein kinase (BCK1) bypass the requirement for a Saccharomyces cerevisiae protein kinase C homolog. 172 97

Monoclonal antibodies (mAbs) to the hinge region of human IgG1 immunoglobulins were prepared by immunization with a proteolytic fragment of hinge segment coupled to keyhole limpet hemocyanin. A mAb, 4G3, was obtained capable of binding to intact IgG but not to partially reduced IgG. Using this mAb, inter-heavy (H) chain disulfide bond formation from partially reduced anti-tetanus antibodies (Abs) in the presence or absence of antigens (Ags) was studied by solid phase radioimmunoassay. When the Abs were partially reduced in solution and then coated to plastic plates, only 10% regeneration of inter-H chain disulfide bonds occurred after reoxidation, although 100% formation occurred in solution [Kishida et al., J. Biochem. (Tokyo) 79, 91-105 (1976)]. This difference in the extent of disulfide bond formation can be explained by the fact that there are two convertible isomers in solution, Conformer I and II, one of which (Conformer I) can form disulfide bonds but is present as a minor component. Since the motion of IgG molecules on plates is restricted by hydrophobic interactions, the two conformers are not convertible as in solution. Therefore only Conformer I which existed before coating formed inter-H chain disulfide bonds. Similar kinetic measurements were performed using plates coated with tetanus toxoid. Abs, partially reduced in solution and then allowed to react with Ags on the plate were able to completely regenerate their inter-H chain disulfide bonds, although the rate of reaction was slow. These results can also be explained by the fact that the two isomers are convertible since Ag-Ab complexes are dissociable. Ag-binding therefore did not significantly perturb conformation of the hinge segments. In addition, no difference between liganded and unliganded Abs was observed in the binding of anti-hinge mAbs. These results imply little or no contribution of the hinge region to transmission of the signal produced by Ag-binding to Fc.
Mol Immunol 1992 Jan
PMID:Kinetics of inter-heavy chain disulfide bond formation of liganded and unliganded human immunoglobulin G by radioimmunoassay. 173 Nov 90

We have isolated and characterized five overlapping clones that encompass 3.2 kb and encode a part of the short subfragment 2, the hinge, and the light meromyosin regions of the myosin heavy chain rod as well as 143 bp of the 3' untranslated portion of the mRNA. Northern blot analysis showed expression of this mRNA mainly in ventricular muscle of the adult chicken heart, with trace levels detected in the atrium. Transient expression was seen in skeletal muscle during development and in regenerating skeletal muscle following freeze injury. To our knowledge, this is the first report of an avian ventricular myosin heavy chain sequence. Phylogenetic analysis indicated that this isoform is a distant homolog of other ventricular and skeletal muscle myosin heavy chains and represents a distinct member of the multigene family of sarcomeric myosin heavy chains. The ventricular myosin heavy chain of the chicken is either paralogous to its counterpart in other vertebrates or has diverged at a significantly higher rate.
J Mol Evol 1991 Oct
PMID:Structural and phylogenetic analysis of the chicken ventricular myosin heavy chain rod. 177 88

Plasmodium vivax and Plasmodium knowlesi merozoites invade only Duffy blood group-positive human erythrocytes. Soluble P. vivax and P. knowlesi merozoite proteins of 135 kDa bind specifically to Duffy blood group determinants. The gene encoding a member of the Duffy receptor gene family of P. knowlesi has been cloned. We report here the molecular cloning of the presumptive Duffy receptor gene of P. vivax, using the P. knowlesi gene as a probe. There is a single gene in P. vivax which codes for a protein of 1115 amino acids. The deduced amino acid sequence predicts a putative signal sequence at the amino-terminus and a transmembrane region followed by 45 amino acids at the carboxy-terminus. The three introns found at the 3' end of the P. knowlesi gene were conserved in P. vivax, including high homology for the sequences of the introns. Comparison of the portion of the proteins amino to the transmembrane region between P. vivax and the partial sequence of P. knowlesi indicated at least three domains. Two homologous regions were separated by a non-homologous region. The cysteines in the homologous regions were conserved in number and position, indicating that the folding is similar and suggesting that these regions may be the Duffy blood group binding domains. In both P. vivax and P. knowlesi, the non-homologous region is hydrophilic and proline-rich, although the position of the prolines is not conserved. As prolines tend to stiffen a protein, this region may act as a 'hinge region' similar to those in the immunoglobulin gene family.
Mol Biochem Parasitol 1991 Jan
PMID:Cloning of the Plasmodium vivax Duffy receptor. 184 31


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