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1.
Proteins ; 60(4): 787-96, 2005 Sep 01.
Article in English | MEDLINE | ID: mdl-16021622

ABSTRACT

The targets of the Structural GenomiX (SGX) bacterial genomics project were proteins conserved in multiple prokaryotic organisms with no obvious sequence homolog in the Protein Data Bank of known structures. The outcome of this work was 80 structures, covering 60 unique sequences and 49 different genes. Experimental phase determination from proteins incorporating Se-Met was carried out for 45 structures with most of the remainder solved by molecular replacement using members of the experimentally phased set as search models. An automated tool was developed to deposit these structures in the Protein Data Bank, along with the associated X-ray diffraction data (including refined experimental phases) and experimentally confirmed sequences. BLAST comparisons of the SGX structures with structures that had appeared in the Protein Data Bank over the intervening 3.5 years since the SGX target list had been compiled identified homologs for 49 of the 60 unique sequences represented by the SGX structures. This result indicates that, for bacterial structures that are relatively easy to express, purify, and crystallize, the structural coverage of gene space is proceeding rapidly. More distant sequence-structure relationships between the SGX and PDB structures were investigated using PDB-BLAST and Combinatorial Extension (CE). Only one structure, SufD, has a truly unique topology compared to all folds in the PDB.


Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli/genetics , Genome, Bacterial , Genomics , Databases, Protein , Enzymes/chemistry , Enzymes/genetics , Escherichia coli Proteins/genetics , Models, Molecular , Protein Conformation , Regression Analysis , X-Ray Diffraction
2.
Structure ; 7(12): 1547-56, 1999 Dec 15.
Article in English | MEDLINE | ID: mdl-10647185

ABSTRACT

BACKGROUND: In Arabidopsis thaliana, ethylene perception and signal transduction into the cell are carried out by a family of membrane-bound receptors, one of which is ethylene resistant 1 (ETR1). The large cytoplasmic domain of the receptor showed significant sequence homology to the proteins of a common bacterial regulatory pathway, the two-component system. This system consists of a transmitter histidine kinase and a response regulator (or signal receiver). We present the crystal structures of the first plant receiver domain ETRRD (residues 604-738) of ETR1 in two conformations. RESULTS: The monomeric form of ETRRD resembles the known structure of the bacterial receiver domain. ETRRD forms a homodimer in solution and in the crystal, an interaction that has not been described previously. Dimerization is mediated by the C terminus, which forms an extended beta sheet with the dimer-related beta-strand core. Furthermore, the loop immediately following the active site adopts an exceptional conformation. CONCLUSIONS: The three-dimensional structure of ETRRD shows the expected conformational conservation to prokaryotic receiver proteins, such as CheY and CheB, both of which are part of the chemotaxis signaling pathway. ETRRD provides the first detailed example of a dimerized receiver domain. Given that the dimer interface of ETRRD coincides with the phosphorylation-dependent interfaces of CheY and CheB, we suggest that the monomerization of ETRRD is phosphorylation-dependent too. In the Mg(2+)-free form of ETRRD, the gamma-loop conformation does not allow a comparable interaction as observed in the active-site architectures of Mg(2+)-bound CheY from Escherichia coli and Salmonella typhimurium.


Subject(s)
Arabidopsis/physiology , Plant Proteins/chemistry , Plant Proteins/physiology , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/physiology , Amino Acid Sequence , Crystallography, X-Ray , Histidine Kinase , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Kinases/metabolism , Protein Structure, Secondary , Sequence Alignment , Sequence Homology, Amino Acid , Signal Transduction , Software
3.
Proc Natl Acad Sci U S A ; 95(26): 15189-93, 1998 Dec 22.
Article in English | MEDLINE | ID: mdl-9860944

ABSTRACT

Many small bacterial, archaebacterial, and eukaryotic genomes have been sequenced, and the larger eukaryotic genomes are predicted to be completely sequenced within the next decade. In all genomes sequenced to date, a large portion of these organisms' predicted protein coding regions encode polypeptides of unknown biochemical, biophysical, and/or cellular functions. Three-dimensional structures of these proteins may suggest biochemical or biophysical functions. Here we report the crystal structure of one such protein, MJ0577, from a hyperthermophile, Methanococcus jannaschii, at 1.7-A resolution. The structure contains a bound ATP, suggesting MJ0577 is an ATPase or an ATP-mediated molecular switch, which we confirm by biochemical experiments. Furthermore, the structure reveals different ATP binding motifs that are shared among many homologous hypothetical proteins in this family. This result indicates that structure-based assignment of molecular function is a viable approach for the large-scale biochemical assignment of proteins and for discovering new motifs, a basic premise of structural genomics.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Carrier Proteins/chemistry , Carrier Proteins/genetics , Genome , Protein Structure, Secondary , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Bacterial Proteins/metabolism , Binding Sites , Carrier Proteins/metabolism , Computer Simulation , Conserved Sequence , Crystallography, X-Ray , Hydrogen Bonding , Methanococcus/genetics , Models, Molecular , Molecular Sequence Data , Sequence Alignment
4.
Acta Crystallogr D Biol Crystallogr ; 54(Pt 4): 690-2, 1998 Jul 01.
Article in English | MEDLINE | ID: mdl-9761877

ABSTRACT

The signal receiver domain of ETR1, an ethylene receptor from Arabidopsis thaliana, has been subcloned and expressed in E. coli and purified by affinity chromatography. Crystals of both native and a selenomethionine-substituted form of the receiver domain have been obtained. Native crystals grew in 1.6 M Li2SO4 and 0.1 M HEPES pH 7. 5 and once flash-frozen diffract to 2.1 A resolution. They belong to space group P41212 with unit-cell dimensions a = b = 48.4, c = 112.3 A.


Subject(s)
Arabidopsis/chemistry , Peptide Fragments/chemistry , Plant Proteins/chemistry , Receptors, Cell Surface/chemistry , Arabidopsis/genetics , Crystallization , Crystallography, X-Ray , DNA, Complementary/genetics , Hydrogen-Ion Concentration , Peptide Fragments/isolation & purification , Plant Proteins/genetics , Plant Proteins/isolation & purification , Protein Structure, Tertiary , Receptors, Cell Surface/genetics , Receptors, Cell Surface/isolation & purification , Recombinant Fusion Proteins/chemistry
5.
Proc Natl Acad Sci U S A ; 93(7): 2735-40, 1996 Apr 02.
Article in English | MEDLINE | ID: mdl-8610110

ABSTRACT

The central role of cyclin-dependent kinases (CDKs) in cell cycle regulation makes them a promising target for studying inhibitory molecules that can modify the degree of cell proliferation. The discovery of specific inhibitors of CDKs such as polyhydroxylated flavones has opened the way to investigation and design of antimitotic compounds. A novel flavone, (-)-cis-5,7-dihydroxyphenyl-8-[4-(3-hydroxy-1-methyl)piperidinyl] -4H-1-benzopyran-4-one hydrochloride hemihydrate (L868276), is a potent inhibitor of CDKs. A chlorinated form, flavopiridol, is currently in phase I clinical trials as a drug against breast tumors. We determined the crystal structure of a complex between CDK2 and L868276 at 2.33 angstroms resolution and refined to an Rfactor 20.3%. The aromatic portion of the inhibitor binds to the adenine-binding pocket of CDK2, and the position of the phenyl group of the inhibitor enables the inhibitor to make contacts with the enzyme not observed in the ATP complex structure. The analysis of the position of this phenyl ring not only explains the great differences of kinase inhibition among the flavonoid inhibitors but also explains the specificity of L868276 to inhibit CDK2 and CDC2.


Subject(s)
CDC2-CDC28 Kinases , Chromones/chemistry , Chromones/pharmacology , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/chemistry , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Piperidines/chemistry , Piperidines/pharmacology , Protein Conformation , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/chemistry , Protein Structure, Secondary , Amino Acid Sequence , Animals , Binding Sites , Cell Line , Crystallography, X-Ray , Cyclin-Dependent Kinase 2 , Cyclin-Dependent Kinases/biosynthesis , Humans , Models, Molecular , Models, Structural , Molecular Sequence Data , Protein Serine-Threonine Kinases/biosynthesis , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Spodoptera , Structure-Activity Relationship , Transfection
6.
Pneumologie ; 49(8): 475-9, 1995 Aug.
Article in German | MEDLINE | ID: mdl-7479643

ABSTRACT

Pneumological examinations including open lung biopsy performed on a male patient of 30 years of age suffering from severe respiratory distress that disabled him, as well as from massive recurring attacks of hemoptysis, resulted in suspicion of idiopathic pulmonary hemosiderosis (also known as Ceelen-Gellerstedt's syndrome). Diagnosis of cor triatriatum followed by surgery was arrived at only after a pulmonary oedema had developed and after other rare cardiac diseases had been considered. This rare congenital malformation--which occasionally becomes clinically manifest only in the adult--should be suspected in differential diagnosis of respiratory distress and a sometimes also life-threatening hemoptysis. Echocardiography is the diagnostic method of choice in this regard.


Subject(s)
Cor Triatriatum/complications , Hemosiderosis/etiology , Lung Diseases, Obstructive/etiology , Lung Diseases/etiology , Adult , Cor Triatriatum/pathology , Cor Triatriatum/surgery , Hemodynamics/physiology , Hemosiderosis/pathology , Hemosiderosis/surgery , Humans , Lung/pathology , Lung Diseases/pathology , Lung Diseases/surgery , Lung Diseases, Obstructive/pathology , Lung Diseases, Obstructive/surgery , Male
7.
J Mol Biol ; 246(4): 522-30, 1995 Mar 03.
Article in English | MEDLINE | ID: mdl-7877173

ABSTRACT

Two crystal structures of ligated uridylate kinase from Saccharomyces cerevisiae were determined by X-ray analyses. The ligands were ADP and AMP. Cocrystallization with ATP yielded crystals with ADP at the ATP site and a mixture of AMP and ADP at the NMP site. Cocrystallization with ADP gave rise to a distinct crystal type with ADP at the ATP site, but only AMP at the NMP site. In both cases, the substrates are kept in place by favorable crystal contacts. The structures have been refined to R-factors of 17.8% and 19.6% at resolutions of 2.1 A and 1.9 A, respectively. A comparison with the related cytosolic adenylate kinase from pig disclosed large induced-fit movements on substrate binding and the disassembly of the catalytic center in the absence of substrates. The relatively high side-activity of uridylate kinase for AMP is explained by the finding that the binding pocket is sized for an AMP, but constructed to bind UMP together with a water molecule.


Subject(s)
Nucleoside-Phosphate Kinase/metabolism , Adenosine Diphosphate/metabolism , Adenosine Monophosphate/metabolism , Amino Acid Sequence , Binding Sites , Catalysis , Crystallization , Models, Molecular , Molecular Sequence Data , Protein Structure, Secondary , Substrate Specificity , Uridine Monophosphate/metabolism
8.
J Mol Biol ; 236(1): 361-7, 1994 Feb 11.
Article in English | MEDLINE | ID: mdl-8107116

ABSTRACT

Uridylate kinase from Saccharomyces cerevisiae is a member of the nucleoside monophosphate (NMP) kinase family and catalyzes the reaction ATP+NMP<==>ADP+NDP with moderate specificity for UMP. The recombinant enzyme crystallized together with two substrate molecules. The structure was solved, by multiple isomorphous replacement and solvent flattening, at 3.0 A and then refined at 2.13 A resolution. The present R-factor is 19%. Superposition onto the structure of a substrate-free adenylate kinase revealed the motions induced by substrate binding. A further superposition onto an adenylate kinase with bound P1,P5-bis(5'-adenosyl)pentaphosphate (Ap5A), a two-substrate-mimicking inhibitor, failed to explain the UMP preference of the uridylate kinase, but superimposed the nucleosides and in particular the non-transferred phosphates at the ATP- and NMP-site rather well. The coincidence of the phosphates indicate strongly that these groups assume their final positions during catalysis. This locates the transition state, which can be modeled with reasonable geometry in agreement with an in-line associative SN2 mechanism.


Subject(s)
Adenosine Diphosphate/metabolism , Nucleoside-Phosphate Kinase/chemistry , Protein Conformation , Saccharomyces cerevisiae/enzymology , Uridine Monophosphate/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Binding Sites , Cloning, Molecular/methods , Escherichia coli , Genes, Fungal , Models, Molecular , Molecular Sequence Data , Nucleoside-Phosphate Kinase/biosynthesis , Nucleoside-Phosphate Kinase/metabolism , Protein Binding , Protein Structure, Secondary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Substrate Specificity , X-Ray Diffraction
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