The three-dimensional structure of ricin at 2.8 A

The x-ray crystallographic structure of the heterodimeric plant toxin ricin has been determined at 2.8-A resolution. The A chain enzyme is a globular protein with extensive secondary structure and a reasonably prominent cleft assumed to be the active site. The B chain lectin folds into two topologically similar domains, each binding lactose in a shallow cleft. In each site a glutamine residue forms a hydrogen bond to the OH-4 of galactose, accounting for the epimerimic specificity of binding. The interface between the A and B chains shows some hydrophobic contacts in which proline and phenylalanine side chains play a prominent role.     

Role of arginine 180 and glutamic acid 177 of ricin toxin A chain in enzymatic inactivation of ribosomes.

The gene for ricin toxin A chain was modified by site-specific mutagenesis to change arginine 180 to alanine, glutamine, methionine, lysine, or histidine. Separately, glutamic acid 177 was changed to alanine and glutamic acid 208 was changed to aspartic acid. Both the wild-type and mutant proteins were expressed in Escherichia coli and, when soluble, purified and tested quantitatively for enzyme activity. A positive charge at position 180 was found necessary for solubility of the protein and for enzyme activity. Similarly, a negative charge with a proper geometry in the vicinity of position 177 was critical for ricin toxin A chain catalysis. When glutamic acid 177 was converted to alanine, nearby glutamic acid 208 could largely substitute for it. This observation provided valuable structural information concerning the nature of second-site mutations.     

Requirements for antiribosomal activity of pokeweed antiviral protein

It has been known for some time that pokeweed antiviral protein acts by enzymatically inhibiting protein synthesis on eucaryotic ribosome systems. The site of this action is known to be the ribosome itself. In this paper we show that the pokeweed antiviral protein reaction against ribosomes is a strong function of salt concentrations, where 160 mM K⁺ and 3 mM Mg²⁺ retards the reaction, while 20 mM K⁺ and 2 mM Mg²⁺ allows maximum reaction rate. It is also shown, however, that an unidentified protein in the postribosomal supernatant solution, together with ATP, allows the ribosome to be attacked even in the presence of high salt. Kinetic analysis of the antiviral protein reaction has been carried out under both sets of conditions, and reveals that the turnover number for the enzyme is about 300–400 mol/mol per min. in each case. The K_m for ribosomes is 1 μM in the presence of low salt and 0.2 μM at higher salt in the presence of postribosomal supernatant factors plus ATP. The antiviral protein reaction is also shown to be pH dependent and is controlled by a residue with pK_a value of approx. 7.0, apparently a histidine. Stoichiometric reaction of the enzyme with iodoacetamide results in a significant loss of antiribosomal activity.     

Structure of a ricin mutant showing rescue of activity by a noncatalytic residue.

Ricin A chain is an N-glycosidase which removes a single adenine base from a conservative loop of 28S rRNA, thereby inactivating eukaryotic ribosomes. The mechanism of action has been proposed to include transition-state stabilization of an oxycarbonium ion on the substrate ribose by interaction with Glu 177. Conversion of Glu 177 to Gln reduces activity nearly 200-fold [Ready, M. P., Kim, Y., & Robertus, J. D. (1991) Proteins: Struct., Funct., Genet. 10, 270-278] while conversion to Ala (E177A) reduces activity only 20-fold [Schlossman, D., Withers, D., Welsh, P., Alexander, A., Robertus, J., & Frankel, A. (1989) Mol. Cell. Biol. 9, 5012-5021]. X-ray analysis of the latter mutant protein shows that a residue at the edge of the active site, Glu 208, rotates into the space left vacant by the mutation. Its rearranged carboxylate partially substitutes for that of Glu 177. This is equivalent to the rescue of enzyme activity by a second-site reversion. Kinetic analysis shows the E177A mutation affects kcat and not Km, consistent with the notion that the carboxylate serves in transition-state stabilization.     

Crystallization of an endochitinase from Hordeum vulgar L. seeds.

Higher plants contain several constitutively expressed proteins for protection against infections by viruses, bacteria and fungi. Here we report the crystallization of a polypeptide with antifungal activity, a 26,000 dalton endochitinase from barley (Hordeum vulgare L.) seeds, in a form suitable for high-resolution X-ray analysis. Crystals were grown by vapor diffusion under several different conditions. The best crystals, obtained with ammonium sulfate as the precipitant, belong to the tetragonal space group P4(1)2(1)2 (P4(3)2(1)2), with cell dimensions a = b = 62.9 A and c = 96.0 A. The cell dimensions are consistent with one endochitinase molecule per asymmetric unit, and the crystals diffract to at least 2.0 A resolution.     

Ricin B chain and discoidin I share a common primitive protein fold

The galactoside-binding B chain of the cytotoxic protein ricin is apparently derived from a conservative exon-sized 40-residue peptide which is repeated four times in the molecule. A very similar peptide can also be seen in the amino acid sequence of the slime mold lectin discoidin I, which itself appears to be the product of a gene duplication. There is presently no chemical or structural evidence concerning the function of this peptide region. Nevertheless, the size of this unit, its prominence in the structure of ricin B chain, and its apparent conservation in carbohydrate-binding proteins from widely divergent organisms suggest that it may represent an extremely ancient galactoside-binding exon unit.     

Lysine 219 participates in NADPH specificity in a flavin-containing monooxygenase from Saccharomyces cerevisiae

The flavin-containing monooxygenase from Saccharomyces cerevisiae (yFMO) uses NADPH and O(2) to oxidize thiol containing substrates such as GSH and thereby generates the oxidizing potential for the ER. The enzyme uses NADPH 12 times more efficiently than NADH. Amino acid sequence analysis suggests that Lys 219 and/or Lys 227 may act as counterions to the 2' phosphate of NADPH and to help determine the preference for pyridine nucleotides. Site directed mutations show that Lys 219 makes the greater contribution to cosubstrate recognition. Conversion of Lys 219 to Ala reduces NADPH dependent activity 90-fold, but has no effect on NADH-dependent activity. Conversion of Lys 227 to Ala reduces NADPH-dependent activity fivefold and NADH-dependent activity threefold. Dissociation constants for NADP(+) to oxidized yFMO were measured spectroscopically. K(d) is 12 microM for the wild-type enzyme and 243 microM for the K219A mutant, consistent with the role of Lys 219 in pyridine nucleotide binding.     

2.8-A crystal structure of a nontoxic type-II ribosome-inactivating protein, ebulin l

Ebulin l is a type-II ribosome-inactivating protein (RIP) isolated from the leaves of Sambucus ebulus L. As with other type-II RIP, ebulin is a disulfide-linked heterodimer composed of a toxic A chain and a galactoside-specific lectin B chain. A normal level of ribosome-inactivating N-glycosidase activity, characteristic of the A chain of type-II RIP, has been demonstrated for ebulin l. However, ebulin is considered a nontoxic type-II RIP due to a reduced cytotoxicity on whole cells and animals as compared with other toxic type-II RIP like ricin. The molecular cloning, amino acid sequence, and the crystal structure of ebulin l are presented and compared with ricin. Ebulin l is shown to bind an A-chain substrate analogue, pteroic acid, in the same manner as ricin. The galactoside-binding ability of ebulin l is demonstrated crystallographically with a complex of the B chain with galactose and with lactose. The negligible cytotoxicity of ebulin l is apparently due to a reduced affinity for galactosides. An altered mode of galactoside binding in the 2gamma subdomain of the lectin B chain primarily causes the reduced affinity.     

The X-ray structure of a chitinase from the pathogenic fungus Coccidioides immitis

The X-ray structure of chitinase from the fungal pathogen Coccidioides immitis has been solved to 2.2 A resolution. Like other members of the class 18 hydrolase family, this 427 residue protein is an eight-stranded beta/alpha-barrel. Although lacking an N-terminal chitin anchoring domain, the enzyme closely resembles the chitinase from Serratia marcescens. Among the conserved features are three cis peptide bonds, all involving conserved active site residues. The active site is formed from conserved residues such as tryptophans 47, 131, 315, 378, tyrosines 239 and 293, and arginines 52 and 295. Glu171 is the catalytic acid in the hydrolytic mechanism; it was mutated to a Gln, and activity was abolished. Allosamidin is a substrate analog that strongly inhibits the class 18 enzymes. Its binding to the chitinase hevamine has been observed, and we used conserved structural features of the two enzymes to predict the inhibitors binding to the fungal enzyme.