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1.
Biochemistry ; 39(44): 13376-82, 2000 Nov 07.
Article in English | MEDLINE | ID: mdl-11063574

ABSTRACT

Src homology 2 (SH2) domains are key modules in intracellular signal transduction. They link activated cell surface receptors to downstream targets by binding to phosphotyrosine-containing sequence motifs. The crystal structure of a Grb2-SH2 domain-phosphopeptide complex was determined at 2.4 A resolution. The asymmetric unit contains four polypeptide chains. There is an unexpected domain swap so that individual chains do not adopt a closed SH2 fold. Instead, reorganization of the EF loop leads to an open, nonglobular fold, which associates with an equivalent partner to generate an intertwined dimer. As in previously reported crystal structures of canonical Grb2-SH2 domain-peptide complexes, each of the four hybrid SH2 domains in the two domain-swapped dimers binds the phosphopeptide in a type I beta-turn conformation. This report is the first to describe domain swapping for an SH2 domain. While in vivo evidence of dimerization of Grb2 exists, our SH2 dimer is metastable and a physiological role of this new form of dimer formation remains to be demonstrated.


Subject(s)
Adaptor Proteins, Signal Transducing , ErbB Receptors/chemistry , Phosphopeptides/chemistry , Proteins/chemistry , Proto-Oncogene Proteins c-met/chemistry , src Homology Domains , Amino Acid Sequence , Conserved Sequence , Crystallization , Crystallography, X-Ray , Dimerization , GRB2 Adaptor Protein , Humans , Macromolecular Substances , Molecular Sequence Data , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Tryptophan/chemistry , Water/chemistry
2.
Nat Struct Biol ; 3(4): 382-7, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8599765

ABSTRACT

The diphtheria tox repressor, DtxR, is a 226 amino acid transition metal ion-activated regulatory protein that controls the expression of diphtheria toxin in toxigenic Corynebacterium diphtheriae. The previously solved three-dimensional DtxR structures have identified two potential metal ion binding sites which may play a role in the activation of DNA binding by the repressor. We have used both X-ray crystallographic and site-directed mutational analysis of DtxR(C102D)-Ni2+ complexes and DtxR to identify the metal ion-binding site which results in the activation of the repressor. We demonstrate that DtxR contains both a primary and an ancillary metal ion binding site. The primary site functions directly in the activation of DNA binding. In contrast, the ancillary site contributes weakly, if at all, to activation.


Subject(s)
Bacterial Proteins/chemistry , Corynebacterium diphtheriae/chemistry , DNA-Binding Proteins/chemistry , Nickel/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Cations, Divalent , Crystallography, X-Ray , DNA Mutational Analysis , DNA, Bacterial/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Models, Molecular , Mutagenesis, Site-Directed , Protein Conformation
3.
Proc Natl Acad Sci U S A ; 92(21): 9843-50, 1995 Oct 10.
Article in English | MEDLINE | ID: mdl-7568230

ABSTRACT

The diphtheria tox repressor (DtxR) of Corynebacterium diphtheriae plays a critical role in the regulation of diphtheria toxin expression and the control of other iron-sensitive genes. The crystal structures of apo-DtxR and of the metal ion-activated form of the repressor have been solved and used to identify motifs involved in DNA and metal ion binding. Residues involved in binding of the activated repressor to the diphtheria tox operator, glutamine 43, arginine 47, and arginine 50, were located and confirmed by site-directed mutagenesis. Previous biochemical and genetic data can be explained in terms of these structures. Conformational differences between apo- and Ni-DtxR are discussed with regard to the mechanism of action of this repressor.


Subject(s)
Apoproteins/chemistry , Bacterial Proteins/chemistry , Corynebacterium diphtheriae/chemistry , DNA-Binding Proteins/chemistry , Nickel/chemistry , Repressor Proteins/chemistry , Amino Acid Sequence , Apoproteins/genetics , Bacterial Proteins/genetics , Binding Sites , Crystallography, X-Ray , DNA-Binding Proteins/genetics , Helix-Turn-Helix Motifs , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Binding , Protein Conformation , Repressor Proteins/genetics , Sequence Homology, Amino Acid
4.
J Mol Biol ; 244(5): 654-6, 1994 Dec 16.
Article in English | MEDLINE | ID: mdl-7990147

ABSTRACT

Crystals of the diphtheria tox repressor (DtxR) from Corynebacterium diphtheriae suitable for structure determination have been obtained. DtxR activated with transition metal ions represses the expression of the structural gene for the diphtheria toxin, tox, which is encoded on the genome of a family of closely related corynebacteriophages. The space group of the obtained crystals is trigonal P3(1)21 or its enantiomorph P3(2)21 with a = b = 64.2 A, c = 220.5 A, alpha = beta = 90 degrees, gamma = 120 degrees. Two monomers comprise the asymmetric unit. The crystals diffract to a resolution of better than 3 A.


Subject(s)
Bacterial Proteins/chemistry , Corynebacterium diphtheriae/chemistry , DNA-Binding Proteins/chemistry , Crystallization , Crystallography, X-Ray
5.
Mol Microbiol ; 14(2): 191-7, 1994 Oct.
Article in English | MEDLINE | ID: mdl-7830565

ABSTRACT

In recent years considerable advances have been made in the understanding of the molecular basis of iron-mediated regulation of diphtheria toxin expression. The tox gene has been shown to be regulated by the heavy metal ion-activated regulatory element DtxR. In the presence of divalent heavy metal ions, DtxR becomes activated and binds to a 9 bp interrupted palindromic sequence. The consensus-binding site has been determined by both the sequence analysis of DtxR-responsive operators cloned from genomic libraries of Corynebacterium diphtheriae as well as by in vitro genetic methods using cyclic amplification of selected targets (CASTing). It is now clear that DtxR functions as a global iron-sensitive regulatory element in the control of gene expression in C. diphtheriae. In addition, the metal ion-activation domain of DtxR is being characterized by both mutational analysis and determination of the X-ray structure at 3.0 A resolution.


Subject(s)
Bacterial Proteins/metabolism , Corynebacterium diphtheriae/genetics , DNA, Bacterial/metabolism , DNA-Binding Proteins/metabolism , Diphtheria Toxin/genetics , Gene Expression Regulation, Bacterial , Iron/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Base Sequence , Binding Sites , Cloning, Molecular , Corynebacterium diphtheriae/growth & development , Corynebacterium diphtheriae/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Diphtheria Toxin/biosynthesis , Genes, Bacterial , Molecular Sequence Data , Operator Regions, Genetic
6.
Nature ; 352(6331): 168-72, 1991 Jul 11.
Article in English | MEDLINE | ID: mdl-2067577

ABSTRACT

Several hundred million tons of toxic mercurials are dispersed in the biosphere. Microbes can detoxify organo-mercurials and mercury salts through sequential action of two enzymes, organomercury lyase and mercuric ion reductase (MerA). The latter, a homodimer with homology to the FAD-dependent disulphide oxidoreductases, catalyses the reaction NADPH + Hg(II)----NADP+ + H+ + Hg(0), one of the very rare enzymic reactions with metal substrates. Human glutathione reductase serves as a reference molecule for FAD-dependent disulphide reductases and between its primary structure and that of MerA from Tn501 (Pseudomonas), Tn21 (Shigella), p1258 (Staphylococcus) and Bacillus, 25-30% of the residues have been conserved. All MerAs have a C-terminal extension about 15 residues long but have very varied N termini. Although the enzyme from Streptomyces lividans has no addition, from Pseudomonas aeruginosa Tn501 and Bacillus sp. strain RC607 it has one and two copies respectively of a domain of 80-85 residues, highly homologous to MerP, the periplasmic component of proteins encoded by the mer operon. These domains can be proteolytically cleaved off without changing the catalytic efficiency. We report here the crystal structure of MerA from the Gram-positive bacterium Bacillus sp. strain RC607. Analysis of its complexes with nicotinamide dinucleotide substrates and the inhibitor Cd(II) reveals how limited structural changes enable an enzyme to accept as substrate what used to be a dangerous inhibitor. Knowledge of the mode of mercury ligation is a prerequisite for understanding this unique detoxification mechanism.


Subject(s)
Bacillus/enzymology , Oxidoreductases/chemistry , Binding Sites , Cadmium/pharmacology , Crystallization , Disulfides/metabolism , Flavin-Adenine Dinucleotide/metabolism , Glutathione Reductase/chemistry , Glutathione Reductase/metabolism , Humans , Macromolecular Substances , Mercury/metabolism , NADP/metabolism , Oxidoreductases/antagonists & inhibitors , Oxidoreductases/metabolism , Protein Conformation
7.
J Biol Chem ; 264(35): 21144-5, 1989 Dec 15.
Article in English | MEDLINE | ID: mdl-2512289

ABSTRACT

NADH peroxidase from Streptococcus faecalis 10C1 has been crystallized from ammonium sulfate solutions using the hanging drop vapor diffusion method. Depending on pH, the crystals grew in the orthorhombic space group I222 or one of its subgroups P222 or P2(1)2(1)2 (or one of its two permutations). In both cases the unit cell axes are a = 76.6 A, b = 132.9 A, and c = 145.7 A. There are two monomers/asymmetric unit in the body-centered crystal form and four in the primitive one. The enzyme is catalytically active in the crystalline state. The crystals diffract to at least 2.5 A resolution; they are stable in the x-ray beam and hence suitable for detailed three-dimensional structure determination.


Subject(s)
Enterococcus faecalis/enzymology , Flavoproteins , Peroxidases , Crystallization , Flavoproteins/isolation & purification , Peroxidases/isolation & purification , Protein Conformation , X-Ray Diffraction
8.
J Biol Chem ; 264(24): 14386-8, 1989 Aug 25.
Article in English | MEDLINE | ID: mdl-2668287

ABSTRACT

The flavoenzyme mercuric ion reductase from Bacillus sp. strain RC607 was purified by dye-ligand affinity chromatography. The protein was crystallized from solutions of high ionic strength, and one of the two crystal forms obtained has proven suitable for x-ray diffraction studies. Preliminary analysis showed that these crystals belong to the tetragonal space group 1422. The unit cell dimensions are a = b = 180.7 A; c = 127.9 A. The diffraction pattern extends to better than 3 A resolution. Crystal density measurements are consistent with one enzyme dimer of 2 x 69,000 Da comprising the asymmetric unit. Trypsin treatment of the native enzyme resulted in the removal of 157 amino acids at the N terminus. After purification, the remaining fragment (amino acids 158-631), which is still fully active in vitro, could be crystallized under the same conditions as native enzyme. Twinning problems, however, did not allow complete analysis of these crystals.


Subject(s)
Bacillus/enzymology , Bacterial Proteins/isolation & purification , Crystallization , Oxidoreductases/isolation & purification , X-Ray Diffraction , Amino Acid Sequence , Escherichia coli/genetics , Genetic Vectors , Hydrolysis , Molecular Weight , Trypsin
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