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Biomedical subjects

W J Cook

Publications and source records attributed to W J Cook.

At least 55 records · Page 3Linked to original sources
PubMed1994

Dissection of the ADR1 protein reveals multiple, functionally redundant activation domains interspersed with inhibitory regions: evidence for a repressor binding to the ADR1c region.

The yeast transcriptional activator ADR1 is required for expression of the glucose-repressible alcohol dehydrogenase gene (ADH2), as well as genes involved in glycerol metabolism. The N-terminal half of the ADR1 protein was shown to contain three separate transactivation domains, including one (TADI) that encompasses the zinc finger DNA-binding domain. While TADII and TADIII were shown to be functionally redundant in activating ADH2 expression, deletion of only TADIII impaired ADR1 control of glycerol metabolism genes. None of these activation domains appeared to be carbon source regulated when separated from the ADH2 promoter context. Interspersed among these activation domains were two regions which, when removed, increased ADR1 activity; one was localized to the site of ADR1c mutations (residues 227 to 239) that allow glucose-insensitive ADH2 expression. The 227-to-239 region blocked ADR1 activity independently of the TAD present on ADR1, ADR1 DNA binding, and specific ADH2 promoter sequences. In addition, this region inhibited the function of a heterologous transcriptional activator. These results are consistent with the existence of an extragenic factor that binds the ADR1c region and represses ADR1 activity and suggest that other factors are responsible for aiding ADR1 in the carbon source regulation of ADH2.

Alcohol Dehydrogenase↗
PubMed1994

Cystic fibrosis transmembrane conductance regulator mutations that disrupt nucleotide binding.

Increasing evidence suggests heterogeneity in the molecular pathogenesis of cystic fibrosis (CF). Mutations such as deletion of phenylalanine at position 508 (delta F508) within the cystic fibrosis transmembrane conductance regulator (CFTR), for example, appear to cause disease by abrogating normal biosynthetic processing, a mechanism which results in retention and degradation of the mutant protein within the endoplasmic reticulum. Other mutations, such as the relatively common glycine-->aspartic acid replacement at CFTR position 551 (G551D) appear to be normally processed, and therefore must cause disease through some other mechanism. Because delta F508 and G551D both occur within a predicted nucleotide binding domain (NBD) of the CFTR, we tested the influence of these mutations on nucleotide binding by the protein. We found that G551D and the corresponding mutation in the CFTR second nucleotide binding domain, G1349D, led to decreased nucleotide binding by CFTR NBDs, while the delta F508 mutation did not alter nucleotide binding. These results implicate defective ATP binding as contributing to the pathogenic mechanism of a relatively common mutation leading to CF, and suggest that structural integrity of a highly conserved region present in over 30 prokaryotic and eukaryotic nucleotide binding domains may be critical for normal nucleotide binding.

Adenosine Triphosphate↗
PubMed1993

Tertiary structures of class I ubiquitin-conjugating enzymes are highly conserved: crystal structure of yeast Ubc4.

The three-dimensional structure of a yeast ubiquitin-conjugating enzyme, encoded by the Saccharomyces cerevisiae UBC4 gene, has been determined at 2.7 A. The structure was solved using molecular replacement techniques and refined by simulated annealing to an R-factor of 0.198. Bond lengths and angles in the molecule have root mean square deviations from ideal values of 0.018 A and 4.0 degrees, respectively. Ubc4 is an alpha/beta protein with four alpha-helices and a four-stranded antiparallel beta-sheet. The ubiquitin-accepting cysteine is located in a cleft between two loops. Comparison with the recently determined structure of a different plant enzyme suggests that class I ubiquitin-conjugating enzymes are highly conserved in their three-dimensional folding. Except for two extra residues at the N- and the C-terminus of the plant enzyme, the C alpha atoms of the two enzymes can be superimposed with a root mean square deviation of only 1.52 A. Greater variations are found between the surfaces of the two molecules, as most of the identical residues between the two enzymes are either buried or clustered on the surface that lies adjacent to the ubiquitin-accepting cysteine. We suggest that this conserved surface functions in protein-protein binding during ubiquitin thiol ester formation.

Amino Acid Sequence↗
PubMed1993

Structure of a sarcoplasmic calcium-binding protein from amphioxus refined at 2.4 A resolution.

The three-dimensional structure of a sarcoplasmic Ca(2+)-binding protein from the protochordate amphioxus has been determined at 2.4 A resolution using multiple-isomorphous-replacement techniques. The refined model includes all 185 residues, three calcium ions, and one water molecule. The final crystallographic R-factor is 0.199. Bond lengths and bond angles in the molecules have root-mean-square deviations from ideal values of 0.015 A and 2.8 degrees, respectively. The overall structure is highly compact and globular with a predominantly hydrophobic core, unlike the extended dumbbell-shaped structures of calmodulin or troponin C. There are four distinct domains with the typical helix-loop-helix Ca(2+)-binding motif (EF hand). The conformation of the pair of EF hands in the N-terminal half of the protein is unusual due to the presence of an aspartate residue in the twelfth position of the first Ca(2+)-binding loop, rather than the usual glutamate. The C-terminal half of the molecule contains one Ca(2+)-binding domain with a novel helix-loop-helix conformation and one Ca(2+)-binding domain that is no longer functional because of amino acid changes. The overall structure is quite similar to a sarcoplasmic Ca(2+)-binding protein from sandworm, although there is only about 12% amino acid sequence identity between them. The similarity of the structures of these two proteins suggests that all sarcoplasmic Ca(2+)-binding proteins will have the same general conformation, even though there is very little conservation of primary structure among the proteins from various species.

Animals↗
PubMed1993

Identification of three genes required for the glucose-dependent transcription of the yeast transcriptional activator ADR1.

Glucose repression of the ADH2 gene from Saccharomyces cerevisiae is mediated by the synthesis and activity of the transcriptional activator ADR1. In this study, we isolated mutations in three new genes (SAF1, SAF2 and SAF3) that suppressed the glucose-insensitive expression of ADH2 caused by the ADR1-5c allele. The mechanism by which the SAF genes maintain ADR1-5c function was investigated. Each of the mutated SAF genes was found to suppress ADR1-5c activity by lowering ADR1-5c steady state mRNA levels 5- to 8-fold under glucose growth conditions. ADR1 mRNA levels were similarly affected by the saf mutations. In contrast, mutations in the SAF genes had little or no effect on ADR1-5c or ADR1 mRNA levels under ethanol growth conditions. The stability of ADR1-5c mRNA was unaffected by mutations in each of the SAF genes, implying that the SAF genes are required for the transcription of ADR1 mRNA under glucose growth conditions. The possible function of the three SAF genes in ADR1 expression is discussed.

Alcohol Dehydrogenase↗
PubMed1993

Musculoaponeurotic fibromatosis (extraabdominal desmoid tumor) in a child with idiopathic multicentric osteolysis.

The fibromatoses are a group of benign proliferations of fibrous tissue with clinical behavior ranging from that of truly malignant tumors to that of benign reactive fibrous proliferations. Some of the superficial fibromatoses are fairly common, but the deep ones, also known as desmoid tumors or musculoaponeurotic fibromatoses, are rare. Idiopathic multicentric osteolysis is a rare skeletal disorder of childhood that causes progressive destruction of bones and renal failure. We recently saw a young girl with both extraabdominal musculoaponeurotic fibromatosis and idiopathic multicentric osteolysis. The classification, diagnosis, and treatment of the deep fibromatoses are reviewed, and the possible association between the minor bone changes occasionally reported in musculoaponeurotic fibromatosis and idiopathic multicentric osteolysis is discussed.

Biopsy↗
PubMed1992

Crystal structure of recombinant human interleukin-4.

The crystal structure of recombinant human interleukin-4 (rhuIL-4) was initially determined at 3.5-A resolution by multiple isomorphous replacement techniques and subsequently refined to a resolution of 2.35 A by simulated annealing. The final crystallographic R-factor, based on all data in the range 6.0-2.35 A (7470 reflections), is 0.232. Bond lengths and bond angles in the molecule have root mean square deviations from ideal values of 0.016 A and 2.4 degrees, respectively. The overall structure is highly compact and globular with a predominantly hydrophobic core. The main structural feature of rhuIL-4 is a four alpha-helix bundle, which composes approximately 58% of the structure. The helices are arranged in a left-handed antiparallel bundle with two overhand connections. Within these connections is a two-stranded antiparallel beta-sheet. Both the tertiary and secondary structures of rhuIL-4 are similar to those of human granulocyte-macrophage colony-stimulating factor. Critical regions for receptor binding are proposed.

Amino Acid Sequence↗
PubMed1992

Structure of a diubiquitin conjugate and a model for interaction with ubiquitin conjugating enzyme (E2).

Covalent ligation of multiubiquitin chains targets eukaryotic proteins for degradation. In such multiubiquitin chains, successive ubiquitins are linked by an isopeptide bond involving the side chain of Lys48 and the carboxyl group of Gly76. The crystal structure of a diubiquitin conjugate has been determined and refined at 2.3-A resolution. The molecule has internal approximate 2-fold symmetry with multiple hydrophobic and hydrophilic contacts along the 2-fold axis. The structure of the diubiquitin conjugate suggests determinants for recognition of multiubiquitin chains. A model for the interaction of diubiquitin and a ubiquitin conjugating enzyme (E2) is proposed.

Amino Acid Sequence↗
PubMed1992

Three-dimensional structure of a ubiquitin-conjugating enzyme (E2).

The x-ray crystal structure of a recombinant ubiquitin-conjugating enzyme (E2) encoded by the UBC1 gene of the plant Arabidopsis thaliana has been determined with the use of multiple isomorphous replacement techniques and refined at 2.4-A resolution by simulated annealing and restrained least-squares. This E2 is an alpha/beta protein, with four alpha-helices and a four-stranded antiparallel beta-sheet. The NH2 and COOH termini, which may be important for interaction with other enzymes and substrates in the ubiquitin-conjugation pathway, are on the opposite side of the molecule from the cysteine residue that binds to the COOH terminus of ubiquitin. This structure should now allow for the rational analysis of E2 function by in vitro mutagenesis and facilitate the effective design of E2s with unique specificities or catalytic functions.

Amino Acid Sequence↗
PubMed1992

Three-dimensional structure of recombinant human granulocyte-macrophage colony-stimulating factor.

The crystal structure of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) has been determined at 2.8 A resolution using multiple isomorphous replacement techniques. There are two molecules in the crystallographic asymmetric unit, which are related by an approximate non-crystallographic 2-fold axis. The overall structure is highly compact and globular with a predominantly hydrophobic core. The main structural feature of rhGM-CSF is a four alpha-helix bundle, which represents approximately 42% of the structure. The helices are arranged in a left-handed antiparallel bundle with two overhand connections. Within the connections is a two-stranded antiparallel beta-sheet. The tertiary structure of rhGM-CSF has a topology similar to that of porcine growth factor and interferon-beta. Most of the proposed critical regions for receptor binding are located on a continuous surface at one end of the molecule that includes the C terminus.

Amino Acid Sequence↗
PubMed1992

Structure of a sarcoplasmic calcium-binding protein from Nereis diversicolor refined at 2.0 A resolution.

The crystal structure of a sarcoplasmic Ca(2+)-binding protein (SCP) from the sandworm Nereis diversicolor has been determined and refined at 2.0 A resolution using restrained least-squares techniques. The two molecules in the crystallographic asymmetric unit, which are related by a non-crystallographic 2-fold axis, were refined independently. The refined model includes all 174 residues and three calcium ions for each molecule, as well as 213 water molecules. The root-mean-square difference in co-ordinates for backbone atoms and calcium ions of the two molecules is 0.51 A. The final crystallographic R-factor, based on 18,959 reflections in the range 2.0 A less than or equal to d less than or equal to 7.0 A, with intensities exceeding 2.0 sigma, is 0.182. Bond lengths and bond angles in the molecules have root-mean-square deviations from ideal values of 0.013 A and 2.2 degrees, respectively. SCP has four distinct domains with the typical helix-loop-helix (EF-hand) Ca(2+)-binding motif, although the second Ca(2+)-binding domain is not functional due to amino acid changes in the loop. The structure shows several unique features compared to other Ca(2+)-binding proteins with four EF-hand domains. The overall structure is highly compact and globular with a predominant hydrophobic core, unlike the extended dumbbell-shaped structure of calmodulin or troponin C. A hydrophobic tail at the COOH terminus adds to the structural stability by packing against a hydrophobic pocket created by the folding of the NH2 and COOH-terminal Ca(2+)-binding domain pairs. The first and second domains show different helix-packing arrangements from any previously described for Ca(2+)-binding proteins.

Animals↗
PubMed1992

Crystallization and preliminary X-ray investigation of a ubiquitin carrier protein (E2) from Arabidopsis thaliana.

Crystals of a recombinant ubiquitin carrier protein from Arabidopsis thaliana have been grown from solutions of ammonium sulfate. The crystals are orthorhombic, space group P2(1)2(1)2(1); the axes are a = 41.8(1) A, b = 44.9(1) A and c = 83.2(1) A. The crystals are quite stable to X-rays and diffract beyond 2.1 A resolution. There is one molecule in the asymmetric unit.

Carrier Proteins↗
PubMed1992

Glucose repression of the yeast ADH2 gene occurs through multiple mechanisms, including control of the protein synthesis of its transcriptional activator, ADR1.

The rate of ADH2 transcription increases dramatically when Saccharomyces cerevisiae cells are shifted from glucose to ethanol growth conditions. Since ADH2 expression under glucose growth conditions is strictly dependent on the dosage of the transcriptional activator ADR1, we investigated the possibility that regulation of the rate of ADR1 protein synthesis plays a role in controlling ADR1 activation of ADH2 transcription. We found that the rate of ADR1 protein synthesis increased 10- to 16-fold within 40 to 60 min after glucose depletion, coterminous with initiation of ADH2 transcription. Changes in ADR1 mRNA levels contributed only a twofold effect on ADR1 protein synthetic differences. The 510-nt untranslated ADR1 mRNA leader sequence was found to have no involvement in regulating the rate of ADR1 protein synthesis. In contrast, sequences internal to ADR1 coding region were determined to be necessary for controlling ADR1 translation. The ADR1c mutations which enhance ADR1 activity under glucose growth conditions did not affect ADR1 protein translation. ADR1 was also shown to be multiply phosphorylated in vivo under both ethanol and glucose growth conditions. Our results indicate that derepression of ADH2 occurs through multiple mechanisms involving the ADR1 regulatory protein.

Alcohol Dehydrogenase↗
PubMed1991

Crystallization and preliminary X-ray investigation of a sarcoplasmic calcium-binding protein from amphioxus.

Crystals of a sarcoplasmic Ca(2+)-binding protein from the protochordate amphioxus have been grown from solutions of ammonium sulfate. The crystals are orthorhombic, space group C222(1), with unit cell axes a = 59.6(1) A, b = 81.3(1) A and c = 82.4(1) A. There is one molecule in the asymmetric unit. The crystals diffract beyond 2.5 A and show less than 20% decline in diffraction intensities after a three day exposure to X-rays from a laboratory rotating anode source.

Ammonium Sulfate↗
PubMed1991

Three-dimensional structure of recombinant human interferon-gamma.

The x-ray crystal structure of recombinant human interferon-gamma has been determined with the use of multiple-isomorphous-replacement techniques. Interferon-gamma, which is dimeric in solution, crystallizes with two dimers related by a noncrystallographic twofold axis in the asymmetric unit. The protein is primarily alpha helical, with six helices in each subunit that comprise approximately 62 percent of the structure; there is no beta sheet. The dimeric structure of human interferon-gamma is stabilized by the intertwining of helices across the subunit interface with multiple intersubunit interactions.

Amino Acid Sequence↗
PubMed1991

Crystallization and preliminary X-ray investigation of recombinant human interleukin 4.

Crystals of recombinant human interleukin 4 have been grown from solutions of ammonium sulfate. The crystals are tetragonal, space-group P4(1)2(1)2 or P4(3)2(1)2; the unit cell axes are a = 92.2(1) A and c = 46.4(1) A. The crystals are stable to X-rays for at least three days and diffract beyond 2.8 A resolution. The crystals contain approximately 63% solvent, assuming there is one molecule in the asymmetric unit.

Humans↗
PubMed1991

Three-dimensional structure of a sarcoplasmic calcium-binding protein from Nereis diversicolor.

The three-dimensional structure of a sarcoplasmic Ca2(+)-binding protein from the sandworm Nereis diversicolor has been determined at 3.0 A resolution using multiple isomorphous replacement techniques. The NH2-terminal half of the molecule contains one variant Ca2(+)-binding domain with a novel helix-loop-helix conformation and one Ca2(+)-binding domain that is no longer functional because of amino acid changes. The overall conformation of this pair of domains is different from any previously described Ca2(+)-binding protein. The COOH-terminal half of the protein contains two Ca2(+)-binding domains with the usual helix-loop-helix configuration and is similar to calmodulin and troponin C. Unlike calmodulin or troponin C, there is no exposed alpha-helix connecting the two halves of the molecule, so the overall structure is much more compact.

Amino Acid Sequence↗