Plasminogen activator inhibitor-1 (PAI-1) is unique among the serine proteinase inhibitors (serpins) in that it can adopt at least three different conformations (active, substrate and latent). We report the X-ray structure of a cleaved substrate variant of human PAI-1, which has a new beta-strand s4A formed by insertion of the amino-terminal portion of the reactive-site loop into beta-sheet A subsequent to cleavage. This is in contrast to the previous suggestion that the non-inhibitory function of substrate-type serpins is mainly due to an inability of the reactive-site loop to adopt this conformation. Comparison with the structure of latent PAI-1 provides insights into the molecular determinants responsible for the transition of the stressed active conformation to the thermostable latent conformation.

Plants developed a diverse battery of defense mechanisms in response to continual challenges by a broad spectrum of pathogenic microorganisms. Their defense arsenal includes inhibitors of cell wall-degrading enzymes, which hinder a possible invasion and colonization by antagonists. The structure of Triticum aestivum xylanase inhibitor-I (TAXI-I), a first member of potent TAXI-type inhibitors of fungal and bacterial family 11 xylanases, has been determined to 1.7-Å resolution. Surprisingly, TAXI-I displays structural homology with the pepsin-like family of aspartic proteases but is proteolytically nonfunctional, because one or more residues of the essential catalytical triad are absent. The structure of the TAXI-I⅐Aspergillus niger xylanase I complex, at a resolution of 1.8 Å, illustrates the ability of tight binding and inhibition with subnanomolar affinity and indicates the importance of the C-terminal end for the differences in xylanase specificity among different TAXI-type inhibitors.

SummaryFructan 1-exohydrolase, an enzyme involved in fructan degradation, belongs to the glycosyl hydrolase family 32. The structure of isoenzyme 1-FEH IIa from Cichorium intybus is described at a resolution of 2.35 Å . The structure consists of an N-terminal fivefold b-propeller domain connected to two C-terminal b-sheets. The putative active site is located entirely in the b-propeller domain and is formed by amino acids which are highly conserved within glycosyl hydrolase family 32. The fructan-binding site is thought to be in the cleft formed between the two domains. The 1-FEH IIa structure is compared with the structures of two homologous but functionally different enzymes: a levansucrase from Bacillus subtilis (glycosyl hydrolase family 68) and an invertase from Thermotoga maritima (glycosyl hydrolase family 32).

Cell-wall invertases play crucial roles during plant development. They hydrolyse sucrose into its fructose and glucose subunits by cleavage of the alpha1-beta2 glycosidic bond. Here, the structure of the Arabidopsis thaliana cell-wall invertase 1 (AtcwINV1; gene accession code At3g13790) is described at a resolution of 2.15 A. The structure comprises an N-terminal fivefold beta-propeller domain followed by a C-terminal domain formed by two beta-sheets. The active site is positioned in the fivefold beta-propeller domain, containing the nucleophile Asp23 and the acid/base catalyst Glu203 of the double-displacement enzymatic reaction. The function of the C-terminal domain remains unknown. Unlike in other GH 32 family enzyme structures known to date, in AtcwINV1 the cleft formed between both domains is blocked by Asn299-linked carbohydrates. A preliminary site-directed mutagenesis experiment (Asn299Asp) removed the glycosyl chain but did not alter the activity profile of the enzyme.

Summary Invertases and fructan exohydrolases (FEHs) fulfil important physiological functions in plants. Sucrose is the typical substrate for invertases and bacterial levansucrases but not for plant FEHs, which are usually inhibited by sucrose. Here we report on complexes between chicory (Cichorium intybus) 1‐FEH IIa with the substrate 1‐kestose and the inhibitors sucrose, fructose and 2,5 dideoxy‐2,5‐imino‐D‐mannitol. Comparisons with other family GH32 and 68 enzyme‐substrate complexes revealed that sucrose can bind as a substrate (invertase/levansucrase) or as an inhibitor (1‐FEH IIa). Sucrose acts as inhibitor because the O2 of the glucose moiety forms an H‐linkage with the acid‐base catalyst E201, inhibiting catalysis. By contrast, the homologous O3 of the internal fructose in the substrate 1‐kestose forms an intramolecular H‐linkage and does not interfere with the catalytic process. Mutagenesis showed that W82 and S101 are important for binding sucrose as inhibitor. The physiological implications of the essential differences in the active sites of FEHs and invertases/levansucrases are discussed. Sucrose‐inhibited FEHs show a Ki (inhibition constant) well below physiological sucrose concentrations and could be rapidly activated under carbon deprivation.

The 1.4 A resolution structure of recombinant mouse tumour-necrosis factor alpha (mTNF) at 100 K has been determined. The crystals are triclinic, space group P1, with unit-cell parameters a = 48.06, b = 48.18, c = 51.01 A, alpha = 114.8, beta = 103.6, gamma = 91.1 degrees. The structure was refined to a final crystallographic R value of 19.7% (Rfree = 23.3%), including 3477 protein atoms, one 2-propanol molecule, one Tris molecule and 240 water molecules. Throughout the crystal lattice, the trimers are differently packed compared with human TNF, which was crystallized in the tetragonal space group P41212 and refined to 2.6 A resolution. The structures of mTNF and human TNF are very similar, diverging mainly in regions that are either flexible and/or involved in crystal packing. Some loops in mTNF which contain residues important for receptor binding are better resolved than in human TNF, such as the surface-exposed loops 30-34 and 144-147, which are also important for receptor specificity. Compared with human TNFs, the channel formed by the three monomers in mTNF is narrower. One 2-propanol molecule trapped in the trimeric channel could be a lead compound for the design of TNF inhibitors.

The molecular structure of itraconazole, C35H38C12-N804, has been determined from single-crystal X-ray diffraction data. The two molecules in the asymmetric unit differ mainly in the conformation of the methoxyt Internal code of the Janssen Research Foundation: R51211.Acta Crystallographica Section C ISSN 0108-2701ISSN 0108- © 1996 2226 C35H38CI2NsO4 phenylpiperazine moiety. Apart from a 180 ° rotation of the triazole ring, the geometry of the dichlorophenylethoxytriazole moiety is almost the same as the dichlorophenylethoxyimidazole geometry found in miconazole, econazole and ketoconazole. CommentItraconazole, (I), is an oral antifungal triazole registered in most countries of the world. In comparison with miconazole and ketoconazole, itraconazole is essentially a more potent antifungal agent with a wider spectrum, a slower clearance and reduced toxicity. All antifungal azoles probably act by an identical mechanism of inhibition of the fungal cytochrome P-450 enzymes. The potency seems to be determined by the affinity and geometric orientation of the nitrogen heterocycle to the heme iron ion, by protonation of the N3 or N4 atom in the imidazole or triazole ring, and by the affinity of the non-ligand portion for the apoprotein of cytochrome P-450 (Vanden Bossche, Marichal, Gorrens, Geerts & Janssen, 1988). The crystal structure of itraconazole has been determined for comparison with the structures of ketoconazole (Peeters, Blaton & De Ranter, 1979a), miconazole (Peeters, Blaton & De Ranter, 1979b) and econazole (Freer, Pearson & Salole, 1986). The asymmetric unit contains two molecules (A and B) partly related by a pseudo-inversion centre at ca 0.25,0.49,0.25 (Fig. 1). The sec-butyl side chain is statistically disordered in both molecules implying that both the R and S configurations are present. The main difference between the two molecules is the orientation of the methoxyphenylpiperazine moiety. In molecule A, the methoxy group is almost perpendicular to the phenyl ring, whereas in molecule B, it is almost coplanar with the phenyl ring. As a consequence, the C70--O71--C72 and O71--C72---C77 angles are enlarged by ca 4 °.Each molecule contains seven rings of which the triazole and benzene rings are essentially planar with a maximum deviation of 0.014 (5) t Partial occupancies (see below). .353 (7) N51--N52 .326 (7) N51---C55 .458 (7) N51--C56 .320 (9) N52---C53 .333 (8) C53--N54 .33 (1) N54----C55 .531 (7) C56--C57 .411 (5) C57--O58 .392 (6) C57--O61 .543 (8) C57--C62 1.444 (7) O58--C59 1.518 (7) C59--C60 1.507 (9) C59---C70 1.423 (6) C60--O61 1.390 (7) C62--C63 1.387 (6) C62--C67 1.395 (8) C63---C64 1.722 (5) C63--C168 1.366 (7) C64--C65 1.377 (8) C65--C66 1.729 (6) C65---C169 1.378 (8) C66--C67 1.426 (6) C70--O71 1.404 (8)2228 C35H38C12N804 N92 -0.9180 (6) 0.2246 (3) 0.1045 (2) N93 -0.9247 (7) 0.2081 (3) 0.1730 (2O71---C72 1.384 (9) C72--C73 1.358 (8) C72---C77 1.382 (9) C73---C74 1.386 (8) C74--C75 1.384 (9) C75--C76 1.427 (8) C75--N78 1.38 ( 1 ) C76--C77 1.452 (7) N78--C79 1.434 (8) N78---C8...

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