The structure of acid protease from Endothia parasitica in cross-linked form at 3.5 A resolution

Electron density map at 3.5 A resolution has been prepared for the strongly cross-linked acid protease from Endothia parasitica with phase angles determined by three uranyl and one platinite derivatives. The mean figure of merit is 0.85. The map fits the known acid protease foldings, and accounts for 326 residues.     

Structure of acid protease from Endothia parasitica in cross-linked form at 2.45-A resolution.

The structure of acid protease from Endothia parasitica in strongly cross-linked form is compared with that of the untreated protein at 2.45-a resolution. The only observed conformation change introduced by the cross-linking reaction is at the N terminal. Otherwise the two main chain structures are essentially identical. Approximately 2 molecules of the inhibitor, 1,2-epoxy-3-(p-nitrophenoxy)propane, are found to be incorporated into each protein molecule. They are covalently bound to the two aspartic residues at the active center.     

Amino acid sequence of endothiapepsin. Complete primary structure of the aspartic protease from Endothia parasitica

The amino acid sequence of endothiapepsin, the aspartic protease from Endothia parasitica has been determined. The enzyme consists of 330 residues. The sequence determination was performed exclusively at the protein level. The homology of this fungal milk-clotting enzyme with aspartic proteases is demonstrated by alignment with pepsin, chymosin, gastricsin, renin, and cathepsin D from various vertebrates and proteinase A from Saccharomyces cerevisiae showing 25-30% identity. The identity with mucor rennin from Mucor pucillus was 21% and with penicillopepsin from Penicillium janthinellum 53%, the fungal enzymes thus representing the lowest as well as the highest degree of homology.     

A structural comparison of 21 inhibitor complexes of the aspartic proteinase from Endothia parasitica.

The aspartic proteinases are an important family of enzymes associated with several pathological conditions such as hypertension (renin), gastric ulcers (pepsin), neoplastic disease (cathepsins D and E), and AIDS (HIV proteinase). Studies of inhibitor binding are therefore of great importance for design of novel inhibitors for potential therapeutic applications. Numerous X-ray analyses have shown that transition-state isostere inhibitors of aspartic proteinases bind in similar extended conformations in the active-site cleft of the target enzyme. Upon comparison of 21 endothiapepsin inhibitor complexes, the hydrogen bond lengths were found to be shortest where the isostere (P1-P'1) interacts with the enzyme's catalytic aspartate pair. Hydrogen bonds with good geometry also occur at P'2, and more so at P3, where a conserved water molecule is involved in the interactions. Weaker interactions also occur at P2, where the side-chain conformations of the inhibitors appear to be more variable than at the more tightly held positions. At P2 and, to a lesser extent, P3, the side-chain conformations depend intriguingly on interactions with spatially adjacent side chains, namely P'1 and P1, respectively. The tight binding at P1-P'1, P3, and P'2 is also reflected in the larger number of van der Waals contacts and the large decreases in solvent-accessible area at these positions, as well as their low temperature factors. Our analysis substantiates earlier proposals for the locations of protons in the transition-state complex. Aspartate 32 is probably ionized in the complexes, its charge being stabilized by 1, or sometimes 2, hydrogen bonds from the transition-state analogues at P1. The detailed comparison also indicates that the P1 and P2 residues of substrate in the ES complex may be strained by the extensive binding interactions at P3, P'1, and P'2 in a manner that would facilitate hydrolysis of the scissile peptide bond.     

Rennin Enzyme of Endothia parasitica

A microbiological screening program was instituted to search for an animal rennet substitute. Among 381 bacteria and 540 fungi tested, only one organism, Endothia parasitica, yielded a suitable enzyme substitute. The fungal rennin enzyme was crystallized and some of its properties were studied. It was found to be water-soluble, nondialyzable, precipitable with (NH(4))(2)SO(4) and organic solvents (e.g., acetone and isopropanol), and destroyed by heating for 5 min at 60 C. It was determined to be most stable in water at pH 4.5 and to have an isoelectric point of pH 5.5. On acid hydrolysis, it yielded: alanine, ammonia, arginine, aspartic acid, cysteic acid, cystine, glutamic acid, glycine, histidine, isoleucine, leucine, phenylalanine, proline, serine, threonine, tyrosine, and valine. No tryptophan was detected after alkaline hydrolysis. Its molecular weight was estimated to be in the range of 34,000 to 39,000. The milk-clotting activities of the fungal and animal rennins proved to be essentially identical in milk containing various concentrations of CaCl(2). Both rennins manifested comparable clotting activities in milk at pH 6.0 to 7.0.     

Terminal structure of hypovirulence-associated dsRNAs in the chestnut blight fungus Endothia parasitica.

The 3'- and 5'-terminal sequences of the five large double-stranded RNA species (L-dsRNA; 4.5-6.0 X 10(6) daltons) of EP713, a hypovirulent strain of Endothia parasitica, were determined by mobility-shift and enzymatic methods. All the L-dsRNAs appeared to have identical terminal sequences. A heteropolymer sequence was found at one 3'-terminus and a poly(A) sequence of variable length at the other. It was possible to label only one 5'-terminus using polynucleotide kinase and [gamma-32P]ATP, and this was shown to be a poly(U) sequence of variable length. We propose that the dsRNAs have the following structure, where X represents a blocking group: (Formula: see text). A recombinant plasmid containing dsRNA-related sequences was constructed. Hybridization analysis using the recombinant probe indicated that the sequence homology among the L-dsRNAs extended beyond these terminal regions and was also shared by small dsRNAs (0.3-0.45 X 10(6) daltons).     

Stimulation of Mycelial Growth of Endothia parasitica by Heavy Metals

Of 16 metal cations tested on agar medium, only copper and iron stimulated mycelial growth of Endothia parasitica in relatively high concentrations. Similarly enhanced growth was produced in high (32%) glucose concentrations and also when the fungus was grown on cellophane placed over the agar surface. E. parasitica secreted large amounts of oxalate that precipitated primarily as calcium oxalate at the periphery of the fungal colony, causing an opaque halo in the medium. Mycelial growth was retarded greatly when calcium oxalate accumulated, but retardation was reversed by copper and iron salts that prevented accumulation of the calcium oxalate crystals. E. parasitica grew well on media containing copper oxalate and copper-calcium oxalate but grew poorly with calcium oxalate as the carbon source and was inhibited by sodium oxalate in the medium. The specificity by which only copper and iron salts stimulated mycelial growth suggested that the metal and oxalate ions interact to form specific oxalate complexes that reverse the inhibition of simple oxalate salts. This probably accounts for enhanced growth in the presence of otherwise toxic levels of metals and oxalate. The stimulation did not occur in liquid cultures.     

Unusual kinetic behavior of Endothia parasitica protease in hydrolysis of small peptides

Endothia parasitica protease hydrolyzes l-leucyl-l-leucine amide and l-leucyl-l-phenylalanine amide at the peptide bond. l-Phenylalanyl-l-leticine amide, N-carbobenzoxy-l-leucyl-l-phenylalanine amide, N-carbobenzoxy-l-leucyl-l-pheml-alanine, N-carbobenzoxy-l-phenylalanyl-l-valine amide, and l-leucyl-β-naphthyl-amide are not hydrolyzed. In contrast to the kinetics of hydrolysis of casein and oxidized B-chain of insulin and activation of trypsinogen by Endothia parasitica protease which are normal, reaction progress curves for hydrolysis of l-leucyl-l-leucine amide and l-leucyl-l-phenylalanine amide are sigrnoidal. Initially, the reaction rates were of the order of 0.5–2.5% of the maximum rates eventually attained. With increasing time of incubation the reaction rates became faster and faster until maximum rates were achieved. This abnormal behavior was not eliminated by recrystallization of substrate or by incubation of enzyme alone or with products of the reaction prior to addition of substrate. Addition of a new aliquot of substrate, vizl-leucyl-l-leucine amide, to the reaction prior to complete hydrolysis of all of a previous aliquot of the same substrate, or reactions containing a mixture of oxidized B chain of insulin and l-leucyl-l-leucine amide, gave normal reaction progress curves. The duration of abnormal behavior before a maximum rate was attained was a function of enzyme concentration and temperature but not of substrate concentration even though substrate was in less than saturating amounts. The reaction data follow second-order autocatalytic kinetics with respect to enzyme concentration. It is proposed that most of the enzyme is in an inactive form in absence of substrate but is rapidly converted to the active form on combination with a good substrate such as trypsinogen, casein, or oxidized B chain of insulin. However, with a poor substrate such as l-leucyl-l-leucine amide, conversion to active enzyme is mediated through formation of an active enzyme-inactive enzyme complex followed by combination with substrate and hydrolysis.     

Crystal structure of the alkaline proteinase Savinase from Bacillus lentus at 1.4 A resolution.

Savinase (EC3.4.21.14) is secreted by the alkalophilic bacterium Bacillus lentus and is a representative of that subgroup of subtilisin enzymes with maximum stability in the pH range 7 to 10 and high activity in the range 8 to 12. It is therefore of major industrial importance for use in detergents. The crystal structure of the native form of Savinase has been refined using X-ray diffraction data to 1.4 A resolution. The starting model was that of subtilisin Carlsberg. A comparison to the structures of the closely related subtilisins Carlsberg and BPN' and to the more distant thermitase and proteinase K is presented. The structure of Savinase is very similar to those of homologous Bacillus subtilisins. There are two calcium ions in the structure, equivalent to the strong and the weak calcium-binding sites in subtilisin Carlsberg and subtilisin BPN', well known for their stabilizing effect on the subtilisins. The structure of Savinase shows novel features that can be related to its stability and activity. The relatively high number of salt bridges in Savinase is likely to contribute to its high thermal stability. The non-conservative substitutions and deletions in the hydrophobic binding pocket S1 result in the most significant structural differences from the other subtilisins. The different composition of the S1 binding loop as well as the more hydrophobic character of the substrate-binding region probably contribute to the alkaline activity profile of the enzyme. The model of Savinase contains 1880 protein atoms, 159 water molecules and two calcium ions. The crystallographic R-factor [formula; see text].     

Two nonhomologus viruses of Cryphonectria (Endothia) parasitica reduce accumulation of specific virulence-associated polypeptides.

Double-stranded RNA responsible for transmissible hypovirulence of Cryphonectria (Endothia) parasitica affected the accumulation of specific polypeptides. Nonhomologous hypovirulence-causing double-stranded RNAs, originating in Europe or North America, affected accumulation of the same polypeptides. Fewer than 5% of detectable proteins were affected, indicating that hypovirulence is probably not the result of general debilitation of the fungus.     

The primary structure of Aspergillus niger acid proteinase A

The complete amino acid sequence of the acid proteinase A, a non-pepsin type acid proteinase from the fungus Aspergillus niger var. macrosporus, was determined by protein sequencing. The enzyme was first dissociated at pH 8.5 into a light (L) chain and a heavy (H) chain, and the L chain was sequenced completely. Further sequencing was performed with the reduced and pyridylethylated or aminoethylated derivative of the whole protein, using peptides obtained by digestions with Staphylococcus aureus V8 protease, trypsin, chymotrypsin, and lysylendopeptidase. The location of the two disulfide bonds was determined by analysis of cystine-containing peptides obtained from a chymotryptic digest of the unmodified protein. These results established that the protein consists of a 39-residue L chain and a 173-residue H chain that associate noncovalently to form the native enzyme of 212 residues (Mr 22,265). This is, to our knowledge, the first time that such a protein with a rather short peptide chain associated noncovalently has been found. No sequence homology is found with other acid or aspartic proteinases, except for Scytalidium lignicolum acid proteinase B, an enzyme unrelated to pepsin by sequence, which has about 50% identity with the present enzyme. These two enzymes, however, are remarkably different from each other in some structural features.     

Three-dimensional structure of proteinase K at 0.15-nm resolution

The crystal and molecular structure of proteinase K was determined by X-ray diffraction data to 0.15-nm resolution. The enzyme belongs to the subtilisin family with an active-site catalytic triad Asp39--His69--Ser224 but is a representative of a subgroup with a free Cys73 close to and 'below' the active His69. Besides this Cys72, proteinase K has two disulfide bonds, Cys34--Cys123 and Cys178--Cys249, which contribute to the stability of the tertiary structure consisting of an extended central parallel beta-sheet decorated by six alpha-helices, three short antiparallel beta-sheets, 18 beta-turns and involving several internal, structurally important water molecules. Proteinase K exhibits two Ca2+-binding sites, one very strong and the other weak, which were the sites of the heavy atoms (Pb2+, Sm3+) used to solve the crystal structure. The weak binding site is liganded to the N and C termini, Thr16 and Asp260, and is only incompletely coordinated by oxygen ligands. The strong binding site is coordinated in the form of a pentagonal bipyramid with the side chain carboxylate of Asp200 and the C = O of Pro175 as apex, and C = O of Val177 and four water molecules in the equatorial plane. Upon removal of this Ca2+, proteinase K loses activity which is interpreted in terms of a local structural deformation involving the substrate-recognition site (Ser132--Gly136), probably associated with a cis----trans isomerization of cis Pro171. Several water molecules are located in the active site. One, W335, is positioned in the 'oxyanion hole' and is displaced by the C = O of the scissile peptide bond of the substrate, as indicated by crystallographic studies with peptide chloromethane inhibitors. Based on these experiments, a reaction mechanism is proposed where the peptide substrate forms a three-stranded antiparallel pleated sheet with the recognition site of proteinase K consisting of Ser132--Leu133--Gly134 on one side and Gly100--Ser101 on the other, followed by expulsion of the oxyanion hole water W335 and hydrolytic cleavage by the Asp39--His69--Serr224 triad. These latter residues display low thermal motion corresponding to well-defined geometry and are hardly accessible to solvent molecules, whereas the recognition-site amino acids are more flexible and partially exposed to solvent.     

New crystal forms and low resolution structure analysis of 20S proteasomes from bovine liver

20S proteasomes from higher eukaryotes have immunological functions rather than those from archibacteria or yeast. To clarify the mechanism of the sorting and production of antigen-presenting peptides, it is important and worthwhile to determine the structure of mammalian proteasomes using a third generation synchrotron radiation source. Here we report new crystal forms of 20S proteasomes from bovine liver and preliminary structure analysis of them. The crystals belong to the same space group but have different cell dimensions. One crystal (form I) belongs to space group P2(1)2(1)2(1) with unit cell dimensions of a = 124.8, b =197.4, c =323.8 A, and diffracts to 3.0 A resolution. The other crystal (form II) belongs to the same space group with a =115.1, b =205.6, c =316. 0 A, and diffracts to 4.0 A resolution. The diffraction data for the form I crystal provided an interpretable electron density map for presenting the structural differences from yeast proteasomes.     

Electron microscopical structure analysis of yeast fatty-acid synthase at low resolution

From an electron microscopical tilt series of the multi-enzyme complex yeast fatty-acid synthase eight three-dimensional molecular structures were obtained by three-dimensional reconstruction of single molecules. The structures confirm present concepts showing a well defined central wall and a sequential arrangement of protein domains in the form of "arches". Additional structural details as ring-shaped parts in the central walls are recognizable. Because of the flattening and the irregular structural deterioration of the single molecules three-dimensional averaging was only partially successful; however, a satisfactory average from five molecules could be obtained. Attempts to find the symmetry of the subunit arrangement by applying correlation methods and by establishing a novel type of correlation analysis ("correlation tables") did not yield a clear proof. However, several strong indications of D3 symmetry were found.     

Purification and characterization of an acid proteinase from mesophilic Mucor sp. solid-state cultures.

The fourth-day extract of a solid-state culture of the mesophilic Mucor sp. (M-105) strain showed a high milk-clotting activity and a clotting/proteolytic activity ratio similar to that of commercial preparations from microbial origin used in cheese manufacture. After ultrafiltration of the crude extract, the milk-clotting proteinase was purified in two steps: ion-exchange followed by size-exclusion chromatography. Enzyme homogeneity was assessed by HPLC, SDS-PAGE and N-terminal residue determination. A pI value of 4.21 was obtained and a molecular weight of 33 kDa was calculated from size-exclusion chromatography and SDS-PAGE data. The optimum pH for proteolytic activity towards dimethylcasein was in the 3.0-3.5 range. The proteinase retained 26 and 13% of its proteolytic activity after a 30-min incubation period, at pH 5.0 and 50 and 60 degrees C, respectively. This evidenced a lower heat stability than that of the thermophilic enzymes currently used in the cheese industry and also than that of bovine chymosin. The enzyme was fully inhibited by pepstatin A and no effect was observed with PMSF, p-CMPS or EDTA. The N-terminal amino acid sequence: GTGTVPVTDDGNLNEYYXTVTVGXP was compared with those from other fungal enzymes.     

Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing.

A method is proposed to restore ab initio low resolution shape and internal structure of chaotically oriented particles (e.g., biological macromolecules in solution) from isotropic scattering. A multiphase model of a particle built from densely packed dummy atoms is characterized by a configuration vector assigning the atom to a specific phase or to the solvent. Simulated annealing is employed to find a configuration that fits the data while minimizing the interfacial area. Application of the method is illustrated by the restoration of a ribosome-like model structure and more realistically by the determination of the shape of several proteins from experimental x-ray scattering data.     

Comparative structure analysis of proteinase inhibitors from the desert locust, Schistocerca gregaria.

The solution structure of three small serine proteinase inhibitors, two natural and one engineered protein, SGCI (Schistocerca gregaria chymotrypsin inhibitor), SGCI[L30R, K31M] and SGTI (Schistocerca gregaria trypsin inhibitor), were determined by homonuclear NMR-spectroscopy. The molecules exhibit different specificities towards target proteinases, where SGCI is a good chymotrypsin inhibitor, its mutant is a potent trypsin inhibitor, and SGTI inhibits both proteinases weakly. Interestingly, SGTI is a much better inhibitor of insect proteinases than of the mammalian ones used in common assays. All three molecules have a similar fold composed from three antiparallel beta-pleated sheets with three disulfide bridges. The proteinase binding loop has a somewhat distinct geometry in all three peptides. Moreover, the stabilization of the structure is different in SGCI and SGTI. Proton-deuterium exchange experiments are indicative of a highly rigid core in SGTI but not in SGCI. We suggest that the observed structural properties play a significant role in the specificity of these inhibitors.     

Crystallographic analysis of the three-dimensional structure of baboon alpha-lactalbumin at low resolution. Homology with lysozyme.

Previous investigations [Jones, Brown, von Glos & Gaunt (1985) Exp. Cell Res. 156, 31-44] have demonstrated the appearance of a new antigenic determinant (recognized by monoclonal antibody 2D6) on the plasma membrane of rat spermatozoa during post-testicular maturation in the epididymis. Identification of the 2D6 antigen on Western blots from one-dimensional SDS/polyacrylamide gels revealed that it co-migrated with a membrane protein (designated Mr 23,000 antigen) present on testicular and immature germ cells, suggesting that one antigen might be a modified version of the other. In the present work, however, we demonstrate that, although they have similar Mr and are present in soluble and membrane-bound forms, the 2D6 and Mr 23,000 antigens are biochemically and immunologically distinct molecules. The properties of the antigens are described and compared. The Mr 23,000 antigen is present on both testicular and cauda epididymidal spermatozoa, has a pI of 6.1, contains no detectable carbohydrate, is not tissue-specific and is degraded by V8 protease. By contrast, the 2D6 antigen is glycosylated, has a broad pI from 4.5 to 6.1, is tissue- and species-specific and is resistant to digestion with V8 protease. Its role in sperm-egg recognition is discussed.