Mutants of the Escherichia coli heat-labile enterotoxin with reduced ADP-ribosylation activity or no activity retain the immunogenic properties of the native holotoxin.

The Escherichia coli heat-labile enterotoxin (LT) is a potent inducer of mucosal immune responses. In a previous study (L. DeHaan, W.

Nitrate binding to Limulus polyphemus subunit type II hemocyanin and its functional implications.

The horseshoe crab, Limulus polyphemus, employs hemocyanin as an oxygen carrier in its hemolymph. This hemocyanin displays cooperative oxygen binding and heterotropic allosteric regulation by protons, chloride ions and divalent cations. Here, we report the crystal structure of Limulus polyphemus subunit type II hemocyanin with a nitrate ion bound in the interface of its first and second domains.

High-resolution structure of the diphtheria toxin repressor complexed with cobalt and manganese reveals an SH3-like third domain and suggests a possible role of phosphate as co-corepressor.

The crystal structure of diphtheria toxin repressor (DtxR) in complex with the corepressor Co2+ has been determined at 2.0 A resolution and in complex with Mn2+ at 2.2 A resolution.

Structure determination of the human protective protein: twofold averaging reveals the three-dimensional structure of a domain which was entirely absent in the initial model.

Mutations in the human 'protective protein' result in the human lysosomal storage disease galactosialidosis. The structure of the human 'protective protein' has been determined using X-ray crystallography to a resolution of 2.2 A.

Structure of the native cysteine-sulfenic acid redox center of enterococcal NADH peroxidase refined at 2.8 A resolution.

In order to obtain the crystal structure of the flavoprotein NADH peroxidase with its native Cys42-sulfenic acid redox center, a strategy combining reduced exposure of crystals to ambient oxygen and data collection at -160 degrees C was applied. The structure of the native enzyme to 2.8 A resolution is described; these results conclusively establish the existence of the Cys42-sulfenic acid as the functional non-flavin redox center of the peroxidase and provide the first structure for any naturally occurring protein-sulfenic acid.

Crystal structure of a new heat-labile enterotoxin, LT-IIb.

Background: Cholera toxin from Vibrio cholerae and the type I heat-labile enterotoxins (LT-Is) from Escherichia coli are oligomeric proteins with AB5 structures. The type II heat-labile enterotoxins (LT-IIs) from E. coli are structurally similar to, but antigenically distinct from, the type I enterotoxins.

Tumor marker disaccharide D-Gal-beta 1, 3-GalNAc complexed to heat-labile enterotoxin from Escherichia coli.

Heat-labile enterotoxin (LT) is part of the cholera toxin (CT) family and consists of a catalytic A subunit and a B pentamer that serves to recognize the oligosaccharide part of the GM1 ganglioside receptor. We report here the crystal structure of heat-labile enterotoxin in complex with the disaccharide portion of the Thomsen-Friedenreich (T-antigen) tumor marker. The toxin:carbohydrate complex is determined to 2.

Protein engineering studies of A-chain loop 47-56 of Escherichia coli heat-labile enterotoxin point to a prominent role of this loop for cytotoxicity.

Heat-labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, is a close relative of cholera toxin (CT). These two toxins share approximately 80% sequence identity, and consists of one 240-residue A chain and five 103-residue B subunits. The B pentamer is responsible for GM1 receptor recognition, whereas the A subunit carries out an ADP-ribosylation of an arginine residue in the G protein, Gs alpha, in the epithelial target cell.

Stabilization of human triosephosphate isomerase by improvement of the stability of individual alpha-helices in dimeric as well as monomeric forms of the protein.

Human triosephosphate isomerase (hTIM) is a dimeric enzyme of identical subunits, adopting the alpha/beta-barrel fold. In a previous work, a monomeric mutant of hTIM was engineered in which Met14 and Arg98, two interface residues, were changed to glutamine. Analysis of equilibrium denaturation of this monomeric mutant, named M14Q/R98Q, revealed that its conformational stability, 2.

Three hTIM mutants that provide new insights on why TIM is a dimer.

Human triosephosphate isomerase (hTIM), a dimeric enzyme, was altered by site-directed mutagenesis in order to determine whether it can be dissociated into monomers. Two hTIM mutants were produced, in which a glutamine residue was substituted for either Met14 or Arg98, both of which are interface residuces. These substitutions strongly interfere with TIM subunit association, since these mutant TIMs appear to exist as compact monomers in dynamic equilibrium with dimers.

Protein-protein interactions in the pyruvate dehydrogenase multienzyme complex: dihydrolipoamide dehydrogenase complexed with the binding domain of dihydrolipoamide acetyltransferase.

Background: The ubiquitous pyruvate dehydrogenase multienzyme complex is built around an octahedral or icosahedral core of dihydrolipoamide acetyltransferase (E2) chains, to which multiple copies of pyruvate decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) bind tightly but non-covalently. E2 is a flexible multidomain protein that mediates interactions with E1 and E3 through a remarkably small binding domain (E2BD).

Results: In the Bacillus stearothermophilus complex, the E2 core is an icosahedral assembly of 60 E2 chains.

Structure of the heat shock protein chaperonin-10 of Mycobacterium leprae.

Members of the chaperonin-10 (cpn10) protein family, also called heat shock protein 10 and in Escherichia coli GroES, play an important role in ensuring the proper folding of many proteins. The crystal structure of the Mycobacterium leprae cpn10 (Ml-cpn10) oligomer has been elucidated at a resolution of 3.5 angstroms.

Crystal structure of recombinant triosephosphate isomerase from Bacillus stearothermophilus. An analysis of potential thermostability factors in six isomerases with known three-dimensional structures points to the importance of hydrophobic interactions.

The structure of the thermostable triosephosphate isomerase (TIM) from Bacillus stearothermophilus complexed with the competitive inhibitor 2-phosphoglycolate was determined by X-ray crystallography to a resolution of 2.8 A. The structure was solved by molecular replacement using XPLOR.

Crystal structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase from Leishmania mexicana: implications for structure-based drug design and a new position for the inorganic phosphate binding site.

The structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the trypanosomatid parasite Leishmania mexicana has been determined by X-ray crystallography. The protein crystallizes in space group P2(1)2(1)2(1) with unit cell parameters a = 99.0 A, b = 126.

Three-dimensional structure of the human 'protective protein': structure of the precursor form suggests a complex activation mechanism.

Background: The human 'protective protein' (HPP) forms a multi-enzyme complex with beta-galactosidase and neuraminidase in the lysosomes, protecting these two glycosidases from degradation. In humans, deficiency of HPP leads to the lysosomal storage disease galactosialidosis. Proteolytic cleavage of the precursor form of HPP involves removal of a 2 kDa excision peptide and results in a carboxypeptidase activity.

Synthesis and structure-activity relationships of analogs of 2'-deoxy-2'-(3-methoxybenzamido)adenosine, a selective inhibitor of trypanosomal glycosomal glyceraldehyde-3-phosphate dehydrogenase.

In continuation of a project aimed at the structure-based design of drugs against sleeping sickness, analogs of 2'-deoxy-2'-(3-methoxybenzamido)adenosine (1) were synthesized and tested to establish structure-activity relationships for inhibiting glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Compound 1 was recently designed using the NAD:GAPDH complexes of the human enzyme and that of Trypanosoma brucei, the causative agent of sleeping sickness. In an effort to exploit an extra hydrophobic domain due to Val 207 of the parasite enzyme, several new 2'-amido-2'-deoxyadenosines were synthesized.

The Arg7Lys mutant of heat-labile enterotoxin exhibits great flexibility of active site loop 47-56 of the A subunit.

The heat-labile enterotoxin from Escherichia coli (LT) is a member of the cholera toxin family. These and other members of the larger class of AB5 bacterial toxins act through catalyzing the ADP-ribosylation of various intracellular targets including Gs alpha. The A subunit is responsible for this covalent modification, while the B pentamer is involved in receptor recognition.

Refined 3.2 A structure of glycosomal holo glyceraldehyde phosphate dehydrogenase from Trypanosoma brucei brucei.

The three-dimensional crystal structure of the enzyme glyceraldehyde phosphate dehydrogenase from the kinetoplastid Trypanosoma brucei brucei has been determined at 3.2 A resolution from a 37% complete data set collected using the Laue method. The crystals used in the structure determination contain one and a half tetrameric enzyme molecules in the asymmetric unit, i.

Surprising leads for a cholera toxin receptor-binding antagonist: crystallographic studies of CTB mutants.

Background: Because agents which inhibit the receptor binding of cholera toxin constitute possible lead compounds for the structure-based design of anti-cholera drugs, detailed investigation of the toxin's receptor-binding site is of key importance. The substitution Gly-->Asp at residue 33 of the cholera toxin B subunit (CTB) has been reported to abolish receptor-binding ability. The substitution Arg35-->Asp has been reported to result in deficient assembly of the AB5 holotoxin.

Crystallographic analyses of NADH peroxidase Cys42Ala and Cys42Ser mutants: active site structures, mechanistic implications, and an unusual environment of Arg 303.

NADH peroxidase from Enterococcus faecalis is a tetrameric flavoenzyme of 201,400 Da which employs Cys 42 as a redox-active center cycling between sulfhydryl (Cys-SH) and sulfenic acid (Cys-SOH) states along the catalytic pathway. The role of the active site cysteine 42 in NADH peroxidase has been elucidated using biochemical and crystallographic techniques. Here we describe the crystal structures of two active site cysteine mutants, Cys42Ala and Cys42Ser, which were determined to 2.

Structural diversity in a conserved cholera toxin epitope involved in ganglioside binding.

Cholera is a widespread disease for which there is no efficient vaccine. A better understanding of the conformational rearrangements at the epitope might be very helpful for the development of a good vaccine. Cholera toxin (CT) as well as the closely related heat-labile toxin from Escherichia coli (LT) are composed of two subunits, A and B, which form an oligomeric assembly AB5.

An L40C mutation converts the cysteine-sulfenic acid redox center in enterococcal NADH peroxidase to a disulfide.

Multiple sequence alignments including the enterococcal NADH peroxidase and NADH oxidase indicate that residues Ser38 and Cys42 align with the two cysteines of the redox-active disulfides found in glutathione reductase (GR), lipoamide dehydrogenase, mercuric reductase, and trypanothione reductase. In order to evaluate those structural determinants involved in the selection of the cysteine-sulfenic acid (Cys-SOH) redox centers found in the two peroxide reductases and the redox-active disulfides present in the GR class of disulfide reductases, NADH peroxidase residues Ser38, Phe39, Leu40, and Ser41 have been individually replaced with Cys. Both the F39C and L40C mutant peroxidases yield active-site disulfides involving the new Cys and the native Cys42; formation of the Cys39-Cys42 disulfide, however, precludes binding of the FAD coenzyme.

Crystallographic and enzymatic investigations on the role of Ser558, His610, and Asn614 in the catalytic mechanism of Azotobacter vinelandii dihydrolipoamide acetyltransferase (E2p).

Dihydrolipoamide acetyltransferase (E2p) is the structural and catalytic core of the pyruvate dehydrogenase multienzyme complex. In Azotobacter vinelandii E2p, residues Ser558, His610', and Asn614' are potentially involved in transition state stabilization, proton transfer, and activation of proton transfer, respectively. Three active site mutants, S558A, H610C, and N614D, of the catalytic domain of A.