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Query: UNIPROT:P06889 (Mol)
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We have developed a new method of detecting common spatial arrangements of backbone fragments in proteins. This method allows corresponding fragments to occur in a different order in respective amino acid sequences. We applied this method to detect structural similarities between an acid protease, endothiapepsin, and all other proteins in the protein data bank. Significant similarities were found not only with other acid proteases but also with virus proteases and with proteins having different functions. The possible biological meaning of these similarities is discussed.
J Mol Biol 1992 May 05
PMID:Common spatial arrangements of backbone fragments in homologous and non-homologous proteins. 158 93

The molecular structure of endothiapepsin (EC 3.4.23.6), the aspartic proteinase from Endothia parasitica, has been refined to a crystallographic R-factor of 0.178 at 2.1 A resolution. The positions of 2389 protein non-hydrogen atoms have been determined and the present model contains 333 solvent molecules. The structure is bilobal, consisting of two predominantly beta-sheet domains that are related by an approximate 2-fold axis. Of approximately 170 residues, 65 are topologically equivalent when one lobe is superimposed on the other. Twenty beta-strands are arranged as five beta-sheets and are connected by regions involving 29 turns and four helices. A central sheet involves three antiparallel strands from each lobe organized around the dyad axis. Each lobe contains a further local dyad that passes through two sheets arranged as a sandwich and relates two equivalent motifs of four antiparallel strands (a, b, c, d) followed by a helix or an irregular helical region. Sheets 1N and 1C, each contain two interpenetrating psi structures contributed by strands c,d,d' and c',d',d, which are related by the intralobe dyad. A further sheet, 2N or 2C, is formed from two extended beta-hairpins from strands b,c and b',c' that fold above the sheets 1N and 1C, respectively, and are hydrogen-bonded around the local intralobe dyad. Asp32 and Asp215 are related by the interlobe dyad and form an intricate hydrogen-bonded network with the neighbouring residues and comprise the most symmetrical part of the structure. The side-chains of the active site aspartate residues are held coplanar and the nearby main chain makes a "fireman's grip" hydrogen-bonding network. Residues 74 to 83 from strands a'N and b'N in the N-terminal lobe form a beta-hairpin loop with high thermal parameters. This "flap" projects over the active site cleft and shields the active site from the solvent region. Shells of water molecules are found on the surface of the protein molecule and large solvent channels are observed within the crystal. There are only three regions of intermolecular contacts and the crystal packing is stabilized by many solvent molecules forming a network of hydrogen bonds. The three-dimensional structure of endothiapepsin is found to be similar to two other fungal aspartic proteinases, penicillopepsin and rhizopuspepsin. Even though sequence identities of endothiapepsin with rhizopuspepsin and penicillopepsin are only 41% and 51%, respectively, a superposition of the three-dimensional structures of these three enzymes shows that 237 residues (72%) are within a root-mean-square distance of 1.0 A.
J Mol Biol 1990 Feb 20
PMID:X-ray analyses of aspartic proteinases. The three-dimensional structure at 2.1 A resolution of endothiapepsin. 217 68

The aspartic proteinase, endothiapepsin (EC 3.4.23.6), was complexed with a highly potent renin inhibitor, H-261 (t-Boc-His-Pro-Phe-His-LeuOHVal-Ile-His), where OH denotes a hydroxyethylene (-(S) CHOH-CH2-) transition-state isostere in the scissile bond surrogate. Crystals were grown in a form that has the same space group P2(1) as the uncomplexed enzyme, but with a 10 A decrease in the length of the alpha-axis and a 13 degrees decrease in the beta-angle. X-ray data have been collected to a resolution of 1.6 A. The rotation and translation parameters defining the position of the enzyme in the unit cell were determined previously using another enzyme-inhibitor complex that crystallized isomorphously with that of H-261. The molecule was refined using restrained least-squares refinement and the positions of non-hydrogen atoms of the inhibitor and water molecules were defined by difference Fourier techniques. The enzyme-inhibitor complex and 322 water molecules were further refined to a crystallographic R-factor of 0.14. Apart from a small rigid group rotation of a domain comprising residues 190 to 302 and small movements in the flap, there is little difference in conformation between the complexed and uncomplexed forms of the enzyme. The inhibitor is bound in an extended conformation along the active site cleft, and the hydroxyl group of the hydroxyethylene moiety is hydrogen-bonded to both catalytic aspartate carboxylates. The complex is stabilized by hydrogen bonds between the main-chain of the inhibitor and the enzyme. All side-chains of the inhibitor are in van der Waals' contact with groups in the enzyme and define a series of specificity pockets along the active site cleft. The study provides useful clues as to how this potent renin inhibitor (IC50 value of 0.7 x 10(-9) M) may bind renin. In particular it defines the interactions of the hydroxyethylene transition-state isostere with the enzyme more precisely than has been previously possible and therefore provides a useful insight into interactions in the transition state complex.
J Mol Biol 1990 Dec 20
PMID:X-ray analyses of aspartic proteinases. III Three-dimensional structure of endothiapepsin complexed with a transition-state isostere inhibitor of renin at 1.6 A resolution. 226 53

A distance-based database search scheme is proposed for modeling Pro----in non-Pro and insertion/deletion regions of homologous globular proteins up to six residues in length. In the first step, geometric descriptors, the number of residues involved and target distances corresponding to the separation of C alpha atom positions adjacent to the "missing" segment, are chosen. In the second step, a database of high-resolution X-ray structures is scanned for segments with similar descriptors and selected segments are binned according to conformational type. In the third and fourth steps, the selected conformations are docked into the protein, and geometric and energetic criteria are used to determine their viability as segment models. The fifth step consists of an interaction scheme in which the geometric descriptors are redefined. This compensates for the use of a limited database and/or for the use of a poor original protein model adjacent to the missing segment. The procedure has been tested on Pro----non-Pro mutations in the homologous proteins penicillopepsin and endothiapepsin, and on the insertion/deletion regions of the homologs penicillopepsin and endothiapepsin, trypsin and gamma-chymotrypsin and hen and human lysozyme. The test cases represent a wide variety of secondary structural elements (helix, sheet, turn and coil) and insertion/deletion lengths (0 to 4 residues). It is shown that 79% of the test cases are accurately modeled (within 0.54 A root-mean-square (r.m.s.) deviation for main-chain atoms) using the proposed scheme. Failure of the scheme (main-chain atom r.m.s. deviations greater than 1.29 A) in 21% of the cases appears to be related to the presence of infrequently observed conformations or locally unique folds of the target proteins with respect to the database (18% of the test cases); the remaining 3% are unexplained. Geometric and energetic criteria are able to discriminate between trial conformations that correspond to the X-ray structures and those that are different in 97% of the conformations generated by the distance-weighted database search scheme. The scheme is shown to be relatively insensitive to uncertainty in the template co-ordinates, since the geometric descriptors were taken from the homologous protein (r.m.s. deviations in the position of descriptors range from 0.18 to 1.35 A for the accurately modeled test cases). It is demonstrated that the scheme can be used to correct local sequence misalignments.
J Mol Biol 1990 Dec 20
PMID:Modeling of globular proteins. A distance-based data search procedure for the construction of insertion/deletion regions and Pro----non-Pro mutations. 226 66

Aspartic proteases (EC3.4.23) are a group of proteolytic enzymes of the pepsin family that share the same catalytic apparatus and usually function in acid solutions. This latter aspect limits the function of aspartic proteases to some specific locations in different organisms; thus the occurrence of aspartic proteases is less abundant than other groups of proteases, such as serine proteases. The best known sources of aspartic proteases are stomach (for pepsin, gastricsin, and chymosin), lysosomes (for cathepsins D and E), kidney (for renin), yeast granules, and fungi (for secreted proteases such as rhizopuspepsin, penicillopepsin, and endothiapepsin). These aspartic proteases have been extensively studied for their structure and function relationships and have been the topics of several reviews or monographs (Tang: Acid Proteases, Structure, Function and Biology. New York: Plenum Press, 1977; Tang: J Mol Cell Biochem 26:93-109, 1979; Kostka: Aspartic Proteinases and Their Inhibitors. Berlin: Walter de Gruyter, 1985). All mammalian aspartic proteases are synthesized as zymogens and are subsequently activated to active proteases. Although a zymogen for a fungal aspartic protease has not been found, the cDNA structure of rhizopuspepsin suggests the presence of a "pro" enzyme (Wong et al: Fed Proc 44:2725, 1985). It is probable that other fungal aspartic proteases are also synthesized as zymogens. It is the aim of this article to summarize the major models of structure-function relationships of aspartic proteases and their zymogens with emphasis on more recent findings. Attempts will also be made to relate these models to other aspartic proteases.
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PMID:Evolution in the structure and function of aspartic proteases. 354 46

A computer procedure TFIT, which uses a molecular superposition force field to flexibly match test compounds to a 3D pharmacophore, was evaluated to find out whether it could reliably predict the bioactive conformations of flexible ligands. The program superposition force field optimizes the overlap of those atoms of the test ligand and template that are of similar chemical type, by applying an attractive force between atoms of the test ligand and template which are close together and of similar type (hydrogen bonding, charge, hydrophobicity). A procedure involving Monte Carlo torsion perturbations, followed by torsional energy minimization, is used to find conformations of the test ligand which cominimize the internal energy of the ligand and the superposition energy of ligand and template. The procedure was tested by applying it to a series of flexible ligands for which the bioactive conformation was known experimentally. The 15 molecules tested were inhibitors of thermolysin, HIV-1 protease or endothiapepsin for which X-ray structures of the bioactive conformation were available. For each enzyme, one of the molecules served as a template and the others, after being conformationally randomized, were fitted. The fitted conformation was then compared to the known binding geometry. The matching procedure was successful in predicting the bioactive conformations of many of the structures tested. Significant deviation from experimental results was found only for parts of molecules where it was readily apparent that the template did not contain sufficient information to accurately determine the bioactive conformation.
J Comput Aided Mol Des 1995 Jun
PMID:Flexible matching of test ligands to a 3D pharmacophore using a molecular superposition force field: comparison of predicted and experimental conformations of inhibitors of three enzymes. 756 76

The discovery that the protease from the human immunodeficiency virus (HIV) belongs to the aspartic protease family has generated renewed interest in this class of proteins. In this paper, the interactions of endothiapepsin, an aspartic proteinase from the fungus Endothia parasitica, with the inhibitor pepstatin A have been studied by high-sensitivity calorimetric techniques. These experiments have permitted a complete characterization of the temperature and pH-dependence of the binding energetics. The binding reaction is characterized by negative intrinsic binding enthalpy and negative heat capacity changes. The association constant is maximal at low pH (2 x 10(9) M-1 at pH 3) but decreases upon increasing pH (8.1 x 10(6) M-1 at pH 7). The binding of the inhibitor is coupled to the protonation of one of the aspartic moieties in the Asp dyad of the catalytic site of the protein. This phenomenon is responsible for the decrease in the apparent affinity of the inhibitor for the enzyme upon increasing pH. The experimental results presented here indicate that the binding of the inhibitor is favored both enthalpically and entropically. While the favorable enthalpic contribution is intuitively expected, the favorable entropic contribution is due to the large gain in solvent-related entropy associated with the burial of a large hydrophobic surface, that overcompensates the loss in conformational and translational/rotational degrees of freedom upon complex formation. The characteristics of the molecular recognition process have been evaluated by means of structure-based thermodynamic analysis. Three regions in the protein contribute significantly to the free energy of binding: the residues surrounding the Asp dyad (Asp32 in the N-terminal lobe and Asp215 in the C-terminal domain) and the flap region (Ile73 to Asp77). In addition, the rearrangement of residues that are not in immediate contact with the inhibitor provides close to 40% of the protease contribution to the binding free energy. On the other hand, the two statine residues provide more than half of the inhibitor contributions to the total free energy of binding. It is demonstrated that a previously developed empirical structural parametrization of the thermodynamic parameters that define the Gibbs energy, accurately accounts for the binding energetics and its temperature and pH-dependence.
J Mol Biol 1995 Sep 22
PMID:Thermodynamic mapping of the inhibitor site of the aspartic protease endothiapepsin. 756 55

The structure of mouse submaxillary renin complexed with a decapeptide inhibitor, CH-66 (Piv-His-Pro-Phe-His-Leu-OH-Leu-Tyr-Tyr-Ser-NH2), where Piv denotes a pivaloyl blocking group, and -OH- denotes a hydroxyethylene (-(S)CHOH-CH2-) transition state isostere as a scissile bond surrogate, has been refined to an agreement factor of 0.18 at 2.0 A resolution. The positions of 10,038 protein atoms and 364 inhibitor atoms (4 independent protein inhibitor complexes), as well as of 613 solvent atoms, have been determined with an estimated root-mean-square (r.m.s.) error of 0.21 A. The r.m.s. deviation from ideality for bond distances is 0.026 A, and for angle distances is 0.0543 A. We have compared the three-dimensional structure of mouse renin with other aspartic proteinases, using rigid-body analysis with respect to shifts involving the domain comprising residues 190 to 302. In terms of the relative orientation of domains, mouse submaxillary renin is closest to human renin with only a 1.7 degrees difference in domain orientation. Porcine pepsin (the molecular replacement model) differs structurally from mouse renin by a 6.9 degrees domain rotation, whereas endothiapepsin, a fungal aspartic proteinase, differs by 18.8 degrees. The triple proline loop (residues 292 to 294), which is structurally opposite the active-site "flap" (residues 72 to 83), gives renin a superficial resemblance to the fold of the retroviral proteinases. The inhibitor is bound in an extended conformation along the active-site cleft, and the hydroxyethylene moiety forms hydrogen bonds with both catalytic aspartate carboxylates. The complex is stabilized by hydrogen bonds between the main chain of the inhibitor and the enzyme. All side-chains of the inhibitor are in van der Waals contact with groups in the enzyme and define ten specificity sub-sites. This study shows how renin has compact sub-sites due to the positioning of secondary structure elements, to complementary substitutions and to the residue composition of its loops close to the active site, leading to extreme specificity towards its prohormone substrate, angiotensinogen. We have analysed the micro-environment of each of the buried charged groups in order to predict their ionization states.
J Mol Biol 1994 Feb 11
PMID:X-ray analysis at 2.0 A resolution of mouse submaxillary renin complexed with a decapeptide inhibitor CH-66, based on the 4-16 fragment of rat angiotensinogen. 810 15

The structure of mucor pusillus pepsin (EC 3.4.23.6), the aspartic proteinase from Mucor pusillus, has been refined to a crystallographic R-factor of 16.2% at 2.0 A resolution. The positions of 2638 protein atoms, 221 solvent atoms and a sulphate ion have been determined with an estimated root-mean-square (r.m.s.) error of 0.15 to 0.20 A. In the final model, the r.m.s. deviation from ideality for bond distances is 0.022 A, and for angle distances it is 0.050 A. Comparison of the overall three-dimensional structure with other aspartic proteinases shows that mucor pusillus pepsin is as distant from the other fungal enzymes as it is from those of mammalian origin. Analysis of a rigid body shift of residues 190 to 302 shows that mucor pusillus pepsin displays one of the largest shifts relative to other aspartic proteinases (14.4 degrees relative to endothiapepsin) and that changes have occurred at the interface between the two rigid bodies to accommodate this large shift. A new sequence alignment has been obtained on the basis of the three-dimensional structure, enabling the positions of large insertions to be identified. Analysis of secondary structure shows the beta-sheet to be well conserved whereas alpha-helical elements are more variable. A new alpha-helix hN4 is formed by a six-residue insertion between positions 131 and 132. Most insertions occur in loop regions: -5 to 1 (five residues relative to porcine pepsin): 115 to 116 (six residues); 186 to 187 (four residues); 263 to 264 (seven residues); 278 to 279 (four residues); and 326 to 332 (six residues). The active site residues are highly conserved in mucor pusillus pepsin; r.m.s. difference with rhizopuspepsin is 0.37 A for 25 C alpha atom pairs. However, residue 303, which is generally conserved as an aspartate, is changed to an asparagine in mucor pusillus pepsin, possibly influencing pH optimum. Substantial changes have occurred in the substrate binding cleft in the region of S1 and S3 due to the insertion between 115 and 116 and the rearrangement of loop 9-13. Residue Asn219 necessitates a shift in position of substrate main-chain atoms to maintain hydrogen bonding pattern. Invariant residues Asp11 and Tyr14 have undergone a major change in conformation apparently due to localized changes in molecular structure. Both these residues have been implicated in zymogen stability and activation.
J Mol Biol 1993 Mar 05
PMID:X-ray analyses of aspartic proteinases. V. Structure and refinement at 2.0 A resolution of the aspartic proteinase from Mucor pusillus. 845 May 40

Two new proteinases secreted by Cryphonectria parasitica, namely EapB and EapC, have been purified. The corresponding structural genes were isolated by screening a cosmid library, and sequenced. Comparison of genomic and cDNA sequences revealed that the eapB and eapC genes contain three and two introns, respectively. The products of the eapB and eapC genes as deduced from the nucleotide sequences, are 268 and 269 residues long, respectively. N-terminal amino acid sequencing data indicates that EapC is synthesized as a zymogen, which yields a mature 206-amino acid enzyme after cleavage of the prepro sequence. Similarly, sequence alignment studies suggest that EapB is secreted as a 203-residue form which shares extensive similarities not only with EapC but also with two other acid fungal proteinases. However, they display distinct structural features; for example, no cysteine residue is found in EapC. The eapC gene was mutated using a two-step gene replacement strategy which allowed the specific introduction of several stop codons at the beginning of the eapC coding sequence in an endothiapepsin-deficient (EapA-) C. parasitica strain. Although the resulting strain did not secrete EapC, it still exhibited residual extracellular proteolytic activity, which could be due to EapB.
Mol Gen Genet 1996 Jan 15
PMID:Cloning and characterization of the eapB and eapC genes of Cryphonectria parasitica encoding two new acid proteinases, and disruption of eapC. 856 93


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