Crystal structures of Escherichia coli glycerol kinase variant S58-->W in complex with nonhydrolyzable ATP analogues reveal a putative active conformation of the enzyme as a result of domain motion.

Escherichia coli glycerol kinase (GK) displays "half-of-the-sites" reactivity toward ATP and allosteric regulation by fructose 1, 6-bisphosphate (FBP), which has been shown to promote dimer-tetramer assembly and to inhibit only tetramers. To probe the role of tetramer assembly, a mutation (Ser58-->Trp) was designed to sterically block formation of the dimer-dimer interface near the FBP binding site [Ormo, M., Bystrom, C., and Remington, S. J. (1998) Biochemistry 37, 16565-16572]. The substitution did not substantially change the Michaelis constants or alter allosteric regulation of GK by a second effector, the phosphocarrier protein IIAGlc; however, it eliminated FBP inhibition. Crystal structures of GK in complex with different nontransferable ATP analogues and glycerol revealed an asymmetric dimer with one subunit adopting an open conformation and the other adopting the closed conformation found in previously determined structures. The conformational difference is produced by a approximately 6.0 degrees rigid-body rotation of the N-terminal domain with respect to the C-terminal domain, similar to that observed for hexokinase and actin, members of the same ATPase superfamily. Two of the ATP analogues bound in nonproductive conformations in both subunits. However, beta, gamma-difluoromethyleneadenosine 5'-triphosphate (AMP-PCF2P), a potent inhibitor of GK, bound nonproductively in the closed subunit and in a putative productive conformation in the open subunit, with the gamma-phosphate placed for in-line transfer to glycerol. This asymmetry is consistent with "half-of-the-sites" reactivity and suggests that the inhibition of GK by FBP is due to restriction of domain motion.

Crystal structure of a complex of Escherichia coli glycerol kinase and an allosteric effector fructose 1,6-bisphosphate.

The three-dimensional structures of Escherichia coli glycerol kinase (GK) with bound glycerol in the presence and absence of one of the allosteric regulators of its activity, fructose 1,6-bisphosphate (FBP), at 3.2 and 3.0 A, are presented. The molecule crystallized in space group P41212, and the structure was solved by molecular replacement. The models were refined with good stereochemistry to final R-factors of 21.1 and 21.9%, respectively. A tetrameric arrangement of monomers was observed which was essentially identical to the proposed inactive tetramer II previously described [Feese, M. D., Faber, H. R., Bystrom, C. E., Pettigrew, D. W., and Remington, S. J. (1998) Structure (in press)]. However, the crystal packing in this form was especially open, permitting the FBP binding site to be occupied and identified. The crystallographic data revealed a most unusual type of FBP binding site formed between two glycine-arginine loops (residues 234-236) where one-half of the binding site is donated by each monomer at the regulatory interface. The molecule of FBP binds in two mutually exclusive modes on a noncrystallographic 2-fold axis at 50% occupancy each; thus, a tetramer of GK binds two molecules of FBP. Ionic interactions between the 1- and 6-phosphates of FBP and Arg 236 were observed in addition to hydrogen bonding interactions between the backbone amide of Gly 234 and the 6-phosphate. No contacts between the protein and the furanose ring were observed. Mutagenesis of Arg 236 to alanine drastically reduced the extent of inhibition of GK by FBP and lowered, but did not eliminate, the ability of FBP to promote tetramer association. These observations are consistent with the previously characterized mechanism of FBP inhibition of GK, in which FBP acts both to promote dimer-tetramer assembly and to inactivate the tetramers.

Glycerol kinase from Escherichia coli and an Ala65-->Thr mutant: the crystal structures reveal conformational changes with implications for allosteric regulation.

BACKGROUND: Glycerol kinase (GK) from Escherichia coli is a velocity-modulated (V system) enzyme that has three allosteric effectors with independent mechanisms: fructose-1,6-bisphosphate (FBP); the phosphocarrier protein IIAGlc; and adenosine nucleotides. The enzyme exists in solution as functional dimers that associate reversibly to form tetramers. GK is a member of a superfamily of ATPases that share a common ATPase domain and are thought to undergo a large conformational change as an intrinsic step in their catalytic cycle. Members of this family include actin, hexokinase and the heat shock protein hsc70. RESULTS: We report here the crystal structures of GK and a mutant of GK (Ala65-->Thr) in complex with glycerol and ADP. Crystals of both enzymes contain the same 222 symmetric tetramer. The functional dimer is identical to that described previously for the IIAGlc-GK complex structure. The tetramer interface is significantly different, however, with a relative 22.3 degrees rotation and 6.34 A translation of one functional dimer. The overall monomer structure is unchanged except for two regions: the IIAGlc-binding site undergoes a structural rearrangement and residues 230-236 become ordered and bind orthophosphate at the tetramer interface. We also report the structure of a second mutant of GK (IIe474-->Asp) in complex with IIAGlc; this complex crystallized isomorphously to the wild type IIAGlc-GK complex. Site-directed mutants of GK with substitutions at the IIAGlc-binding site show significantly altered kinetic and regulatory properties, suggesting that the conformation of the binding site is linked to the regulation of activity. CONCLUSIONS: We conclude that the new tetramer structure presented here is an inactive form of the physiologically relevant tetramer. The structure and location of the orthophosphate-binding site is consistent with it being part of the FBP-binding site. Mutational analysis and the structure of the IIAGlc-GK(IIe474-->Asp) complex suggest the conformational transition of the IIAGlc-binding site to be an essential aspect of IIAGlc regulation.

Amplificación de oncogenes en cancer de mama: amplificación del oncogen neu en carcinomas y del gen del receptor del factor de crecimiento epidérmico (EGF-r) en un sarcoma filodes / Oncogene amplification in breast cancer: new amplification in carcinoma and EGF-r amplification in a phyllodes sarcoma

En el presente trabajo se aborda el estudio de 51 tumores primarios humanos de la mama, con el objeto de conocer si existen patrones de amplificación o de reordenamiento en nueve genes de factores de crecimiento o de sus receptores, algunos de los cuales son oncogenes. Los resultados obtenidos mediante la técnica del Southern blot evidenciaron amplificación grosera del oncogene neu en 6 de 42 carcinomas y del gen del EGF-r en un sarcoma filoides. Se verificó el polimorfismo del gen del EGF-r reportado con la enzima Bgl II. No se encontraron evidencias de alteraciones en ninguno de los restantes genes estudiados (EGF, TGF alfa, PDGF-A, PDGF-B. PDGF-R. IGF-I, IGF-II). Se discute el papel de la amplificación de los oncogenes en el desarrollo de las neoplasias (AU)

Amplificación de oncogenes en cancer de mama: amplificación del oncogen neu en carcinomas y del gen del receptor del factor de crecimiento epidérmico (EGF-r) en un sarcoma filodes / Oncogene amplification in breast cancer: new amplification in carcinoma and EGF-r amplification in a phyllodes sarcoma

En el presente trabajo se aborda el estudio de 51 tumores primarios humanos de la mama, con el objeto de conocer si existen patrones de amplificación o de reordenamiento en nueve genes de factores de crecimiento o de sus receptores, algunos de los cuales son oncogenes. Los resultados obtenidos mediante la técnica del Southern blot evidenciaron amplificación grosera del oncogene neu en 6 de 42 carcinomas y del gen del EGF-r en un sarcoma filoides. Se verificó el polimorfismo del gen del EGF-r reportado con la enzima Bgl II. No se encontraron evidencias de alteraciones en ninguno de los restantes genes estudiados (EGF, TGF alfa, PDGF-A, PDGF-B. PDGF-R. IGF-I, IGF-II). Se discute el papel de la amplificación de los oncogenes en el desarrollo de las neoplasias

Genomic alterations in human breast carcinomas.

All human chromosomes were screened in 52 human breast carcinomas for the occurrence of allele losses, in order to identify genomic alterations involved in initiation and progression of the disease. Loss of chromosome 22 alleles was detected in 6 out of 8 lobular carcinomas, while chromosome 17 losses were most frequent in ductal carcinomas. Furthermore, patients who developed advanced disease after many years of mild clinical course showed significantly higher frequencies of allele losses in their primary tumors, compared to patients with a persistently mild disease course. Finally, in one case, molecular examination suggested a translocation t(10;17) with coamplification of the ERBB2 oncogene and chromosome 10 sequences present in the two tumors from this patient, consistent with one of the tumors being a metastasis originating from a subclone of cells in the other tumor.

Localization of the MEN1 gene to a small region within chromosome 11q13 by deletion mapping in tumors.

The gene for multiple endocrine neoplasia type 1 (MEN1), an inherited predisposition to neuroendocrine neoplasm of the parathyroid glands, the pancreatic islet parenchyma, and the anterior pituitary gland, was recently mapped to chromosome 11q13 based on genetic linkage in families. We now show that the pathogenesis of MEN1-associated parathyroid lesions involves unmasking of a recessive mutation at the disease locus and that sporadic primary hyperparathyroidism shares the same mechanisms. By examination of allele losses in MEN1-associated lesions, we could define deletions of chromosome 11 and map the MEN1 locus to a small region within chromosome band 11q13, telomeric to the PYGM locus. In contrast, a low incidence of deletions involving the MEN1 gene was found in sporadic pituitary adenomas.

Localization of the genetic defect in multiple endocrine neoplasia type 1 within a small region of chromosome 11.

Multiple endocrine neoplasia type I (MEN-1), a Mendelian disorder with an autosomal dominant mode of inheritance, causes hyperplasia in the parathyroid glands and hyperplasia or neoplasm in the anterior pituitary gland and/or the pancreatic islets. The genetic defect responsible for MEN-1 in three families was recently mapped to the long arm of chromosome II by linkage between the MEN-1 locus and the gene for skeletal muscle glycogen phosphorylase (PYGM) at 11q13. We have constructed a genetic linkage map of seven markers in the vicinity of the MEN-1 locus that has allowed us to map more precisely the gene associated with MEN-1; the target region has been narrowed to about 12 cM. The closely linked markers will be useful also for identification of likely carriers in families in which an allele responsible for MEN-1 segregates.