Trypsin activity reduced by an autocatalytically produced nonapeptide.

Trypsin, a serine protease enzyme plays a pivotal role in digestion and is autocatalytic. The crystal structure of a complex formed between porcine trypsin and an auto catalytically produced peptide is reported here. This complex shows a reduction in enzyme activity as compared to native beta-trypsin. The nonapeptide has a lysine, which is recognized by Asp 189 at the specificity pocket. The auto catalytically produced native nonapeptide is bound at the active site cleft like other trypsin inhibitors but the important interactions with the oxyanion hole are absent. The peptide covers only a part of the active site cleft and hence the enzyme activity is reduced rather than being inhibited.

Structures of porcine beta-trypsin-detergent complexes: the stabilization of proteins through hydrophilic binding of polydocanol.

Polydocanol has a wide range of medical applications, especially in sclerotherapy of many diseases such as gastrointestinal antiplastia, oesophageal haemangioma etc. It is of interest to study the mode of binding of this medically important detergent and its subsequent action on proteins. Here, three crystal structures of serine protease trypsin are reported in the presence of varying concentrations of polydocanol in order to elucidate its mode of binding and interactions with proteins. Polydocanol binds to the protein with its hydrophilic head rather than the hydrophobic tail as is the case with other detergents such as SDS and MEGA-8. This hydrophilic binding mode results in the binding sites of polydocanol being distributed on the surface of the enzyme. There are at least 11 binding sites for polydocanol in trypsin. Polydocanol forms part of the large-scale water networks which connect distant regions of the enzyme, thereby stabilizing it. The hydrophilic binding of polydocanol also results in cross-linked pairs of trypsin molecules.

Conformational effects of C(alpha,alpha)-dipropargylglycine as a constrained residue.

A useful synthon to approach artificial phenylalanyl peptides in a [2 + 2 + 2] cycloaddition reaction, C(alpha,alpha)-dipropargylglycine (Dprg) is examined for its conformational preferences as a constrained residue. Crystal structure analysis and preliminary NMR results establish possible preference of the residue for folded (alpha) rather than extended (beta) region of the straight phi,psi conformational space. Boc-Dprg-L-Leu-OMe (1) displays two molecular conformations within the same crystallographic asymmetric unit, with Dprg in the alpha(R) or alpha(L) conformation, participating in a type I beta-turn or an alpha(L)-alpha(R)-type fold, in which Leu(2) assumes the alpha(R) conformation stereochemically favored for an L-chiral residue. Boc-Dprg-D-Val-L-Leu-OMe (2) displays a type I' beta-turn conformation in crystal, with both Dprg(1) and D-Val(2) assuming the alpha(L) conformation stereochemically favored for a D-chiral residue, with 4 --> 1 type hydrogen bond linking L-Leu(3) NH with Boc CO. NMR analysis using temperature variation, solvent titration, and a spin probe study suggests a fully solvent-exposed nature of Dprg NH, ruling out a fully extended C(5)-type conformation for this residue, and solvent sequestered nature of L-Leu(3) NH, suggesting possibility of a beta-turn due to Dprg assuming a folded conformation.

Conformational preferences of heterochiral peptides. Crystal structures of heterochiral peptides Boc-(D) Val-(D) Ala-Leu-Ala-OMe and Boc-Val-Ala-Leu-(D) Ala-OMe--enhanced stability of beta-sheet through C-H...O hydrogen bonds.

The crystal structures of Boc-(D) Val-(D) Ala-Leu-Ala-OMe (vaLA) and Boc-Val-Ala-Leu-(D) Ala-OMe (VALa) have been determined. vaLA crystallises in space group P2(1),2(1),2(1), with a = 9.401 (4), b = 17.253 (5), c = 36.276 (9)A. V = 5,884 (3) A3, Z = 8, R = 0.086. VALa crystallises in space group P2(1) with a = 9.683 (9), b = 17.355 (7), c = 18.187 (9) A, beta = 95.84 (8) degrees , V = 3,040(4) A3, Z = 4, R = 0.125. There are two molecules in the asymmetric unit in antiparallel beta-sheet arrangement in both the structures. Several of the Calpha hydrogens are in hydrogen bonding contact with the carbonyl oxygen in the adjacent strand. An analysis of the observed conformational feature of D-chiral amino acid residues in oligopeptides, using coordinates of 123 crystal structures selected from the 1998 release of CSD has been carried out. This shows that all the residues except D-isoleucine prefer both extended and alphaL conformation though the frequence of occurence may not be equal. In addition to this, D-leucine, valine, proline and phenylalanine have assumed alphaR conformations in solid state. D-leucine has a strong preference for helical conformation in linear peptides whereas they prefer an extended conformation in cyclic peptides.

Purification, crystallisation and preliminary X-ray study of haemoglobin from Crocodilis palustris and Crocodilis porosus.

The unsolved three-dimensional structure of crocodile haemoglobin and its prospects as a blood substitute have led us to initiate the purification and crystallisation of haemoglobin molecules from crocodile species (Crocodilis palustris or mugger and Crocodilis porosus or salt water crocodile). The work has resulted in the prevention of polymerisation of naked haemoglobin molecules using N-ethylmaleimide or iodoacetamide. The purified monomeric haemoglobin molecule of C. porosus was crystallised in two different forms and X-ray diffraction data were collected up to 2 A resolution for both forms. Form I: a=53.62, b=53.55, c=103.77 A; beta=93.35 degrees, space group P2(1), Z=2. Form II: a=71.30, b=54.70, c=80.00 A; beta=106.4 degrees, space group P2(1), Z=2. Structure solution and rigid body refinement of form I data resulted in a model with R(free)=0.42 and R=0.35.

Crystal structure at 1.63 A resolution of the native form of porcine beta-trypsin: revealing an acetate ion binding site and functional water network.

The active center of a serine protease is the catalytic triad composed of His-57, Ser-195 and Asp-102. The existing crystal structure data on serine proteases have not fully answered a number of fundamental questions relating to the catalytic activity of serine proteases. The new high resolution native porcine beta-trypsin (BPT) structure is aimed at extending the knowledge on the conformation of the active site and the ordered water structure within and around the active site. The crystal structure of BPT has been determined at 1.63 A resolution. An acetate ion bound at the active site of a trypsin molecule by both classical hydrogen bonds and C-HellipsisO hydrogen bonds has been identified for the first time. A large network of water molecules extending from the recognition amino acid Asp-184 to the entry of the active site has been observed in the BPT structure. A detailed comparison with inhibitor complexes and autolysates indicates that the sulfate ion and the acetate ion bind at the same site of the trypsin molecule. The Ser-195 Cbeta-Ogamma-His-57 Nepsilon angle in the catalytic triad of BPT is intermediate between the corresponding values of the complex and native structure due to acetate ion binding. The network of waters from the recognition amino acid to the active site entry is probably the first ever complete picture of functional waters around the active site. Structural comparisons show that the functional waters involved in the binding of small molecule inhibitors and protease inhibitors are distinctly different.

1-Isobutyl-2,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-3-indoleacetic acid.

The title compound (C17H25NO3) is a perhydroindoleacetic acid which exhibits hypoglycemic activity. The cyclohexyl ring adopts a sofa conformation. The molecule contains an intramolecular O-H...O hydrogen bond.

The structure and function of antiamoebin I, a proline-rich membrane-active polypeptide.

BACKGROUND: Antiamoebin is a member of the peptaibol family of polypeptides and has a unique antibiotic activity: it acts as an antiamoebic agent, but does not effectively haemolyze erythrocytes even though it does exhibit membrane-modifying activity. RESULTS: The structure of antiamoebin I has been determined by X-ray crystallography at 1.4 A resolution. The molecule forms a helical structure, which, as a result of the presence of a number of proline and hydroxyproline residues, has a deep bend in the middle. Circular dichroism spectroscopy, single-channel conductance studies and fluorescence diffusion studies suggest a mode of ion transport that is entirely different from that of the other two members of the peptaibol family (alamethicin and zervamicin) whose structures and functions have been examined in detail. CONCLUSIONS: The structure of the polypeptide has been determined and a functional model for its mode of action in membranes is presented. Although under some conditions antiamoebin may form ion channels, unlike the closely related alamethicin and zervamicin polypeptides, its major membrane-modifying activity appears to be as an ion carrier.

Structural basis for selective inhibition of COX-2 by nimesulide.

Nimesulide 1 is a novel nonsteroidal antiinflammatory drug which inhibits the enzyme cyclooxygenase 2 (COX-2) more selectively than cyclooxygenase 1 (COX-1). Molecular modelling studies have been carried out on complexes of 1 with COX-1 and with mutants of COX-1 simulating COX-2. These indicate that the mutations I523V and S516A largely contribute to the selectivity. A comparative study with SC-558 2 has also been performed.

Reengineering a type II beta-turn as a potential helix nucleator. Part I. Crystal structure of Boc-Val-Pro-(D)Asp-Asp-Val-OMe monohydrate.

The crystal structure of Boc-Val1-Pro2-(D)Asp3-Asp4-Val5-OMe is described as a type II beta-turn reengineered into a potential helix nucleator. (D)Asp3 in the peptide is responsible for the configurationally guided LD chiral type II beta-turn centered at Pro2-(D)Asp3, as well as the partially developed LL chiral type I beta-turn centered at Asp4-Val5 by acceptance of a conformation nucleating H-bond from Val5NH to its carboxylic oxygen.

The first structure at 1.8 A resolution of an active autolysate form of porcine alpha-trysoin.

The first crystal structure of an active autolysate form of porcine alpha-trypsin (APT), a two-chain molecule obtained from the limited autolysis of porcine beta-trypsin at position Lys145-Ser146, has been determined. APT crystallizes in space group P2(1)2(1)2(1) with one protein molecule in the asymmetric unit. The structure was solved by molecular replacement followed by refinement using X-PLOR to an R factor of 0.200 and an R(free) of 0.285 for 8.0-1.8 A data with r.m.s deviations from ideal values of 0.01 A and 1.7 degrees for bond lengths and bond angles, respectively. Comparison with inactive autolysate porcine epsilon-trypsin (EPT) and porcine beta-trypsin in complex with bittergourd trypsin inhibitor (MCT) revealed a small but systematic directional chain shift around the active-site residues from APT to EPT to MCT.

C-H...O hydrogen bonds in beta-sheets.

A detailed analysis of the occurrence of the C-H...O hydrogen bonds in sheet regions of proteins has been presented. 11 unique protein structures with resolution 1.3 A containing beta-sheets show a widespread presence of C-H...O hydrogen bonds. These have average C(alpha).O, CH...O distances and a C(alpha)-H...O angle of 3.29, 2.38 A and 143 degrees, respectively. As in the case of N-H...O hydrogen bonds, parallel and antiparallel beta-sheet regions show the same hydrogen-bond geometry. An inverse correlation is observed between the hydrogen-bond geometries involving the C(alpha)(i)-H...O=C and the N(i+1)-H...O=C suggesting that C-H...O hydrogen bonds may act as an additional stabilizing factor. The propensity of different amino-acid residues to form such hydrogen bonds varies and shows a clear preference for valine and threonine. C-H...O hydrogen bonds involving side chains also occur extensively in beta-sheet regions.

Crystal structures of heterochiral peptides. Part II. tert-Boc-valyl-D-alanyl-leucyl-alanyl methoxide.

Crystal structure of a heterochiral peptide, viz. Boc-Val1-D-Ala2-Leu3-Ala4-OMe with a D chiral residue in the second position of a sequence, has been determined [a = 40.44(1), b = 4.887(5), c = 15.381(5) A, beta = 109.6(1)degrees, space group C2, Z = 4, R = 0.11]. The peptide is in a parallel beta-sheet structure terminated by a distinct local bend. The structure is established by N-H...O as well as C alpha-H...O hydrogen bonds. The contiguous C alpha-H...O hydrogen bond observed in this structure is an unique observation.

Crystal structures of a D-residue containing tetrapeptides. 1. tert-Boc-D-valyl-alanyl-leucyl-alanyl-methoxide, butanol solvate.

The crystal structure of a heterochiral peptide, viz. Boc-D-Val-Ala-Leu-Ala-OMe, with a D-residue in the beginning of the sequence has been determined (a = 9.464(5), b = 35.615(5), c = 9.703(2) A, space group P2(1)2(1)2, Z = 4, R = 0.09). The peptide is in the extended beta-conformation and the packing is stabilised by four N--H ... O hydrogen bonds in an antiparallel beta-sheet arrangement. The solvent molecule is disordered and does not have any specific interactions with the peptide.

Biological action of lantadene C, a new hepatotoxicant from Lantana camara var. aculeata.

Lantadene C (22 beta-2-methylbutanoyloxy-3-oxoolean-12-en-28-oic acid) isolated from the leaves of the hepatotoxic plant Lantana camara var. aculeata (Red) has been found to be identical with dihydrolantadene A reported earlier. Molecular structure of lantadene C has been deduced from single crystal X-ray diffraction analysis. It resembles lantadene A in the pentacyclic portion of the molecule but differs in the side chain region. Atom C-34 is cis to C-35 in lantadene C but is trans in lantadene A. Semisynthetic lantadene C was prepared by catalytic hydrogenation of lantadene A. Lantadene C was obtained in two forms, I and II. Form I was crystalline while form II was amorphous. Unlike lantadene A, both form I and II of lantadene C elicited strong hepatotoxic response in guinea pigs associated with decrease in fecal output, feed intake, hepatomegaly, hepatic injury at the cellular and subcellular level, increase in plasma bilirubin, and acid phosphatase activity. All the clinical signs, hepatic lesions, and changes in blood plasma typified lantana toxicity. This is the first report on the hepatoxicity of lantadene C. The interrelation of molecular structure and biological activity of lantadene A and C has been discussed.

Molecular structure, polymorphism, and toxicity of lantadene A, the pentacyclic triterpenoid from the hepatotoxic plant Lantana camara.

Lantadene A (22 beta-angeloyloxy-3-oxo-olean-12-en-28-oic acid), a pentacyclic triterpenoid compound from lantana (Lantana camara) leaves has been obtained in two polymorphic forms I and II. Form I had white, fluffy, and rod-shaped uniform crystals. Form II particles were irregular, shining, and polyhedral. The two forms differed in melting behavior. The powder x-ray diffraction of form I showed sharp peaks whereas from II did not contain distinct peaks. From single-crystal three-dimensional x-ray structure determination, the molecular structure of form I has been established. A/B and B/C rings of the molecule are trans fused while D/E rings are cis fused. The packing of the molecule is stabilized by hydrogen bonding. Form I of lantadene A was non-toxic to guinea pigs on oral administration. Form II induced ictericity and toxicity associated with decrease in feed intake and fecal output, hepatomegaly, increase in plasma bilirubin, and acid phosphatase activity.

Structure of methyl 5-phenyl-2-propionyl-3-pyrrolecarboxylate.

C15H15NO3, Mr = 257.4, monoclinic, P2(1)/n, a = 10.070 (2), b = 5.335 (3), c = 24.23 (1) A, beta = 91.60 (3) degrees, V = 1301 (2) A3, Z = 4, Dm (flotation) = 1.312 (3), Dx = 1.314 g cm-3, Cu K alpha (lambda = 1.5418 A), mu = 6.64 cm-1, F(000) = 544, T = 295 K, final R(F) = 0.037, wR = 0.035 for 850 significant reflections, I greater than or equal to 2.5 sigma(I). The phenyl ring and the pyrrole ring are planar, and the dihedral angle between them is 18.6 degrees. The nitrogen of the pyrrole ring exhibits N-H tautomerism and forms N-H...O type hydrogen bonds with the screw related carbonyl oxygen. In the unit cell the molecules form dimers across the centres of inversion and infinite spirals across the screw axis.

Structure of a 3 alpha-(D-methylglycoside) of 7,8 beta-epoxysinogenin--a cardioactive steroid.

C30H42O10, Mr = 562.66, orthorhombic, P2(1)2(1)2(1), a = 10.125 (1), b = 15.632 (2), c = 36.115 (4) A, V = 5716 (1) A3, Z = 8, Dm = 1.312 (2) (flotation), D chi = 1.308 g cm-3, Cu K alpha(lambda = 1.5418 A), mu = 7.18 cm-1, F(000) = 2416, T = 295 K. Final R(F) = 0.07 for 3348 significant reflections with I greater than or equal to 2.5 sigma(I). The A, B, C, D rings of the aglycone ring are found to be in cis-trans-cis fashion forming a buckled structure. The lactone is in C17 beta conformation. The molecules are stabilized by intermolecular hydrogen bonds. The longest direction of the steroid molecule is nearly parallel to the a axis. The conformational features exhibited by the molecule support proposals on activity.

Iron-sulfur clusters in Azotobacter ferredoxin at 2.5 A resolution.

X-ray crystallographic study of the ferredoxin-like protein (iron-sulfur protein III) from Azotobacter vinelandii has been extended to 2.5-A resolution. A 4.0-A resolution electron density map revealed that the molecule contains two Fe-S clusters of differing size and shape separated by 12 A (Stout, C.D. (1979) Nature 279, 83-84). Recent Mössbauer results by Emptage et al. (Emptage, M.H., Kent, T.A., Huynh, B.H., Rawlings, J., Orme-Johnson, W.H., and Münck, E. (1980) J. Biol. Chem. 255, 1793-1796) have shown that the molecule contains 7 iron atoms as a high potential iron protein-like Fe4 center and a novel 3-Fe center. The 2.5-A electron density map shows two distinctly different Fe-S clusters. The larger cluster consists of a tetranuclear [4Fe-4S] core ligated to the protein at each iron atom. The smaller cluster is distinctly planar and cannot be modeled with [2Fe-2S] or [4Fe-4S] structures. The best model for this cluster is a [3Fe-3S] core. The protein makes six contacts with this cluster.