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Query: UNIPROT:P06889 (Mol)
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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 intron of the Rhizopus aspartic proteinase gene (RNAP-I) was modified by in vitro mutagenesis and examined for its splicing efficiency in Saccharomyces cerevisiae. The wild-type intron of the RNAP-I gene was not spliced at all in spite of its structural similarity to introns of S. cerevisiae. The primary transcript of the RNAP-I gene was converted to correctly translatable mRNA only when the complete consensus sequence of S. cerevisiae introns (i.e. 5'-GTATGT-----TACTAAC-----TAG-3') was introduced into its intron, although the efficiency of splicing was low. It is also shown that transformants carrying the RNAP-I gene with the complete consensus sequence of S. cerevisiae introns produce active RNAP-I protein.
Mol Gen Genet 1990 Aug
PMID:Correct splicing of modified introns of a Rhizopus proteinase gene in Saccharomyces cerevisiae. 225 33

A detailed and rule-based side-chain modelling procedure for globular proteins is presented. It uses the conformational information contained in a homologous (template) structure as a starting point and includes recipes for atom placement and for checking and improving the atomic positions. The scheme does not rely on intuitive judgements or visual examination of the model during construction or refinement. It comprises four stages; the first three are relatively simple and the fourth is more complex. In the first stage, initial conformations for as many atoms as possible are transferred from the template structure based on the application of trends reported previously. Second, these trends are used to correct poor van der Waals overlaps. Third, the remaining side-chains atoms (those for which no information is contained in the template) are placed by evaluating their rigid rotation, van der Waals surfaces. The fourth stage consists of a hierarchial series of conformational checks. They involve the evaluation of individual residue energies in the absence and presence of the rest of the protein relative to statistical trends observed in the template structure, the comparison of hydrogen-bonding patterns and side-chain accessibilities in the model and template and brief energy minimization followed by an evaluation of the rigid rotation potential energy surfaces of each side-chain. The checks pinpoint "incorrectly" modelled side-chains, suggest conformational changes and provide a means for determining the portions of the model that are likely to be correct and those likely to be in error. The procedure developed in the paper is tested by modelling the side-chains of the C-terminal lobe of the aspartyl proteinase rhizopuspepsin, using the rhizopuspepsin backbone and the homologous protein, penicillopepsin, as a template for the side-chains. The resultant model was compared to the high-resolution X-ray structure of rhizopuspepsin. Using penicillopepsin data only (stage I), 58% of the chi 1 dihedrals and 44% of the chi 2 dihedrals were modelled correctly. Once poor van der Waals overlaps had been corrected and all of the atoms had been placed (stages II and III), 86% of the chi 1 dihedrals and 75% of the chi 2 dihedrals were correct. After the refinement had been completed (stage IV), 92% of the chi 1 dihedrals and 81% of the chi 2 dihedrals were correctly positioned.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1989 Dec 20
PMID:Construction of side-chains in homology modelling. Application to the C-terminal lobe of rhizopuspepsin. 269 42

The structure of rhizopuspepsin (EC 3.4.23.6), the aspartic proteinase from Rhizopus chinensis, has been refined to a crystallographic R-factor of 0.143 at 1.8 A resolution. The positions of 2417 protein atoms have been determined with a root-mean-square (r.m.s.) error of 0.12 A. In the final model, the r.m.s. deviation from ideality for bond distances is 0.010 A, and for angle distances it is 0.034 A. During the course of the refinement, a calcium ion and 373 water molecules, of which 17 are internal, have been located. The active aspartate residues, Asp35 and Asp218, are involved in similar hydrogen-bonding interactions with neighboring residues and with several water molecules. One water molecule is located between the two carboxyl groups of the catalytic aspartate residues in a tightly hydrogen-bonded position. The refinement resulted in an unambiguous interpretation of the highly mobile "flap", a beta-hairpin loop region that projects over the binding pocket. Large solvent channels are formed when the molecules pack in the crystal, exposing the binding pocket and making it easily accessible. Intermolecular contacts involve mainly solvent molecules and a few protein atoms. The three-dimensional structure of rhizopuspepsin closely resembles other aspartic proteinase structures. A detailed comparison with the structure of penicillopepsin showed striking similarities as well as subtle differences in the active site geometry and molecular packing.
J Mol Biol 1987 Aug 20
PMID:Structure and refinement at 1.8 A resolution of the aspartic proteinase from Rhizopus chinensis. 331 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

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