ABSTRACT
In order to develop a more complete understanding of urea induced protein denaturation we have investigated the crystal structure of urea with the cyclic dipeptide diketopiperazine. This structure, determined to an R factor of 8.1%, shows extensive hydrogen bonding between urea and the peptide groups of diketopiperazine. These studies support a model where hydrogen bonding plays an important contribution in urea-induced protein denaturation. In the companion paper we present thermodynamic data for urea-peptide interactions in aqueous solution that further support this model.
Subject(s)
Piperazines/chemistry , Urea/chemistry , Crystallization , Diketopiperazines , Dipeptides/chemistry , Hydrogen Bonding , Models, Molecular , Molecular Structure , Protein DenaturationABSTRACT
Pseudomonas aeruginosa exotoxin A is a representative of a class of enzymes, the mono-ADP-ribosyl transferases, which catalyze the covalent transfer of an ADP-ribose moiety of NAD+ to a target substrate. Availability of the three-dimensional structure of exotoxin A provides the opportunity for mapping substrate binding sites and suggesting which amino acid residues may be involved in catalysis. Data from several sources have been combined to develop a proposal for the NAD+ binding site of exotoxin A: the binding of NAD+ fragments adenosine, AMP, and ADP have been delineated crystallographically to 6.0, 6.0, and 2.7 A, respectively; significant sequence homology spanning 60 residues has been found between exotoxin A and diphtheria toxin, which has the identical enzymatic activity; iodination of exotoxin A, under conditions in which only tyrosine 481 is iodinated in the enzymatic domain, abolishes ADP-ribosyl transferase activity.
Subject(s)
ADP Ribose Transferases , Bacterial Toxins , Exotoxins/analysis , Peptide Mapping , Pseudomonas aeruginosa/enzymology , Virulence Factors , Amino Acid Sequence , Binding Sites , Crystallography , Diphtheria Toxin/analysis , Fourier Analysis , Iodine , Ligands , Models, Molecular , Molecular Sequence Data , NAD/metabolism , Tetranitromethane , Pseudomonas aeruginosa Exotoxin AABSTRACT
Exotoxin A of Pseudomonas aeruginosa is a secreted bacterial toxin capable of translocating a catalytic domain into mammalian cells and inhibiting protein synthesis by the ADP-ribosylation of cellular elongation factor 2. The protein is a single polypeptide chain of 613 amino acids. The x-ray crystallographic structure of exotoxin A, determined to 3.0-A resolution, shows the following: an amino-terminal domain, composed primarily of antiparallel beta-structure and comprising approximately half of the molecule; a middle domain composed of alpha-helices; and a carboxyl-terminal domain comprising approximately one-third of the molecule. The carboxyl-terminal domain is the ADP-ribosyltransferase of the toxin. The other two domains are presumably involved in cell receptor binding and membrane translocation.
Subject(s)
ADP Ribose Transferases , Bacterial Toxins , Carrier Proteins , Exotoxins , Nucleotidyltransferases , Pseudomonas aeruginosa , Receptors, Cell Surface , Virulence Factors , Biological Transport , Enzyme Precursors , Models, Molecular , Poly(ADP-ribose) Polymerases , Protein Conformation , Receptors, Cholinergic/metabolism , Solubility , X-Ray Diffraction , Pseudomonas aeruginosa Exotoxin AABSTRACT
The protective antigen protein, one of the three separate proteins constituting the exotoxin system of Bacillus anthracis, has been crystallized in a form suitable for structural studies. The crystal form which is most amenable to x-ray analysis is orthorhombic, space group P2(1)2(1)2(1), a = 101.1 A, b = 95.4 A, c = 87.3 A, with one protective antigen monomer/asymmetric unit. The crystals diffract to approximately 3.0-A resolution.
