Rational design, synthesis, and biological activity of benzoxazinones as novel factor Xa inhibitors.

Inappropriate thrombus formation within blood vessels is the leading cause of mortality in the industrialized world. Factor Xa (FXa) is a trypsin-like serine protease that plays a key role in the blood coagulation cascade and represents an attractive target for anticoagulant drug development. From a high-throughput in vitro mass screen of our chemical library, we identified 4-[5-[(2R,6S)-2, 6-dimethyltetrahydro-1(2H)-pyridinyl]pentyl]-2-phenyl-2H-1, 4-benzoxazin-3(4H)-one (1a) as an inhibitor of FXa with an IC(50) of 27 microM. Through a combination of SAR studies and molecular modeling, we synthesized 3-(4-[5-[(2R,6S)-2, 6-dimethyltetrahydro-1(2H)-pyridinyl]pentyl]-3-oxo-3,4-dihydro-2H- 1,4-benzoxazin-2-yl)-1-benzenecarboximidamide (1n) which was a potent FXa inhibitor with an IC(50) of 3 nM. This compound exhibited high selectivity for FXa over other related serine proteases and was efficacious when dosed intravenously in rabbit and dog antithrombotic models.

[Color Doppler imaging of central retinal artery in retinopathy of prematurity].

Color Doppler imaging (CDI) is a noninvasive technique, combining 2-dimensional brightness-modulated (B-mode) ultrasound evaluation of eye and orbital structures, with simultaneous color-coded Doppler imaging of orbital blood flow. It has been used to characterize various ophthalmic disorders in adults. Currently there is no data describing orbital blood flow parameters in either normal children or in those with ophthalmic disease, such as the retinopathy of prematurity (ROP). We evaluated blood flow in the central retinal artery of preterm infants undergoing examination for ROP. We also investigated whether useful readings could be obtained on a consistent basis, and the reproducibility of differences in central retinal artery blood flow between subjects with and without ROP (including the influence of "plus" disease). We obtained hemodynamic readings in 43 of 46 eyes of preterm infants. 13 eyes had no signs of ROP; 18 had ROP (at least stage 1) without "plus" disease, and 12 had ROP with "plus" disease. There were no statistically significant differences in systolic blood flow velocity within the 3 groups. However the average velocity was slower in the "plus" disease group, correlating with the clinical finding of dilated and tortuous blood vessels which characterize the posterior retina of ROP eyes with "plus" disease.

Structural basis of the thrombin selectivity of a ligand that contains the constrained arginine mimic (2S)-2-amino-(3S)-3-(1-carbamimidoyl- piperidin-3-yl)-propanoic acid at P1.

We have studied the thrombin and trypsin complexed structures of a pair of peptidomimetic thrombin inhibitors, containing different P1 fragments. The first has arginine as its P1 fragment, and the second contains the constrained arginine mimic (2S)-2-amino-(3S)-3-(1-carbamimidoyl-piperidin-3-yl)-propano ic acid (SAPA), a fragment known to enhance thrombin/trypsin selectivity of inhibitors. On the basis of an analysis of the nonbonded interactions present in the structures of the trypsin and thrombin complexes of the two inhibitors, the calculated accessible surfaces of the enzymes and inhibitors in the four complexes, data on known structures of trypsin complexes of inhibitors, and factor Xa inhibitory potency of these compounds, we conclude that the ability of this arginine mimic to increase thrombin selectivity of an inhibitor is mediated by its differential interaction with the residue at position 192 (chymotrypsinogen numbering). Thrombin has a glutamic acid at residue 192, and trypsin has a glutamine. The analysis also suggests that this constrained arginine mimic, when present in an inhibitor, might enhance selectivity against other trypsin-like enzymes that have a glutamine at residue position 192.

Color doppler imaging of the central retinal artery in premature infants undergoing examination for retinopathy of prematurity.

PURPOSE: Recent attempts have been made to quantify blood flow velocity in the central retinal artery (CRA) of adults using color Doppler imaging (CDI). Although retinal vascular abnormalities are the hallmark of severe retinopathy of prematurity (ROP), normal values have not been established for CRA blood flow velocity in premature infants. METHODS: CDI of the CRA was successfully performed on 43 eyes in 22 infants (postconceptional ages 32 to 39 weeks) before the infants underwent examination for ROP. Peak systolic velocity (PSV) and end diastolic velocity were recorded from at least 1 eye of each patient. Pourcelot's resistive index was then calculated for each eye studied. RESULTS: Mean PSV for patients with no ROP (n = 6) was 7.2 +/- 1.5 cm/s, whereas those with any degree of ROP excluding plus disease (n = 9) had a mean PSV of 8.9 +/- 1.8 cm/s. Of the patients with ROP and plus disease (n = 7), the mean PSV was 7.0 +/- 1.6 cm/s. There were no statistically significant differences among these 3 groups (P= .08). CONCLUSIONS: CDI can be successfully performed on preterm infants and yields values lower than those previously reported in healthy adult subjects. PSV in the CRA may be higher in subjects with ROP in the absence of plus disease; however, further study is needed to determine whether these differences are significant.

Potent and selective bicyclic lactam inhibitors of thrombin: Part 3: P1' modifications.

The synthesis and antithrombotic activity of a series of nonpeptide bicyclic thrombin inhibitors are described. We have explored the SAR around the P1' site. Modification of the P1' site has been found to affect potency and selectivity.

Mechanism of triclosan inhibition of bacterial fatty acid synthesis.

Triclosan is a broad-spectrum antibacterial agent that inhibits bacterial fatty acid synthesis at the enoyl-acyl carrier protein reductase (FabI) step. Resistance to triclosan in Escherichia coli is acquired through a missense mutation in the fabI gene that leads to the expression of FabI[G93V]. The specific activity and substrate affinities of FabI[G93V] are similar to FabI. Two different binding assays establish that triclosan dramatically increases the affinity of FabI for NAD+. In contrast, triclosan does not increase the binding of NAD+ to FabI[G93V]. The x-ray crystal structure of the FabI-NAD+-triclosan complex confirms that hydrogen bonds and hydrophobic interactions between triclosan and both the protein and the NAD+ cofactor contribute to the formation of a stable ternary complex, with the drug binding at the enoyl substrate site. These data show that the formation of a noncovalent "bi-substrate" complex accounts for the effectiveness of triclosan as a FabI inhibitor and illustrates that mutations in the FabI active site that interfere with the formation of a stable FabI-NAD+-triclosan ternary complex acquire resistance to the drug.

Design of peptidomimetics that inhibit the association of phosphatidylinositol 3-kinase with platelet-derived growth factor-beta receptor and possess cellular activity.

Phosphorylated tyrosine residues of growth factor receptors that associate with intracellular proteins containing src-homology 2 (SH2) domains are integral components in several signal transduction pathways related to proliferative diseases such as cancer, atherosclerosis, and restenosis. In particular, a phosphorylated pentapeptide [pTyr751-Val-Pro-Met754-Leu (pTyr = phosphotyrosine)] derived from the primary sequence of platelet-derived growth factor-beta (PDGF-beta) receptor blocks the association of the C-terminal SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) to PDGF-beta receptor with an IC50 of 0.445 +/- 0.047 microM. Further evaluation of the structure-activity relationships for pTyr751-Val-Pro-Met-Leu resulted in the design of smaller peptidomimetics with enhanced affinity including Ac-pTyr-Val-Ala-N(C6H13)2 (IC50 = 0.076 +/- 0.010 microM). In addition, the phosphotyrosine residue was replaced with a difluorophosphonate derivative [4-phosphono(difluoromethyl)phenylalanine (CF2Pmp)] which has been shown to be stable to cellular phosphatases. The extracellular administration of either CF2Pmp-Val-Pro-Met-Leu or Ac-CF2Pmp-Val-Pro-Met-NH2 in a whole cell assay resulted in specific inhibition of the PDGF-stimulated association from the C-terminal SH2 domain of the p85 subunit of PI 3-kinase to the PDGF-beta receptor in a dose-dependent manner. These compounds were also effective in inhibiting GLUT4 translocation, c-fos expression, and cell membrane ruffling in single-cell microinjection assay.

Potent and selective bicyclic lactam inhibitors of thrombin: Part 2: P1 modifications.

The synthesis and antithrombotic activity of a series of nonpeptide bicyclic thrombin inhibitors is described. We have explored the SAR with modifications to the P1 site. The introduction of arginine mimetics at the P1 site led to potent and selective thrombin inhibitors.

Design, synthesis, and cocrystal structure of a nonpeptide Src SH2 domain ligand.

The specific association of an SH2 domain with a phosphotyrosine (pTyr)-containing sequence of another protein precipitates a cascade of intracellular molecular interactions (signals) which effect a wide range of intracellular processes. The nonreceptor tyrosine kinase Src, which has been associated with breast cancer and osteoporosis, contains an SH2 domain. Inhibition of Src SH2-phosphoprotein interactions by small molecules will aid biological proof-of-concept studies which may lead to the development of novel therapeutic agents. Structure-based design efforts have focused on reducing the size and charge of Src SH2 ligands while increasing their ability to penetrate cells and reach the intracellular Src SH2 domain target. In this report we describe the synthesis, binding affinity, and Src SH2 cocrystal structure of a small, novel, nonpeptide, urea-containing SH2 domain ligand.

Unilateral keratoconus. Incidence and quantitative topographic analysis.

OBJECTIVE: This study was designed to determine the incidence of unilaterality in a population of patients with clinical keratoconus and to compare quantitative descriptors of topography between affected corneas from patients with unilateral keratoconus and corneas of patients with bilateral disease. DESIGN: Retrospective clinical study with new evaluation of some patients. PARTICIPANTS: One hundred sixty-four patients from the University of Texas South-western Medical Center and Wills Eye Hospital who were diagnosed to have moderate-to-advanced keratoconus on the basis of characteristic topographic patterns associated with corneal thinning. INTERVENTION: Corneal topography was evaluated in both eyes of each patient. MAIN OUTCOME MEASURES: Quantitative descriptors of corneal topography were compared between 5 affected corneas of patients with unilateral keratoconus (combined cases from University of Texas Southwestern, LSU Eye Center, and Wills Eye Hospital) and 15 corneas of patients with moderate-to-advanced bilateral keratoconus (1 cornea from each patient). Indices selected for statistical analysis were the Keratoconus Predication Index (KPI), Surface Asymmetry Index (SAI), and Surface Regularity Index (SRI). RESULTS: Three (1.83%) of 164 patients with moderate-to-advanced keratoconus had no topographic evidence of keratoconus in the opposite eye. There were no statistically significant differences in KPI, SRI, or SAI values between the affected corneas of patients with unilateral and bilateral keratoconus. Over a period of 4 years of observation, signs of keratoconus in the previously normal eye developed in a patient with unilateral keratoconus. CONCLUSIONS: The authors found no differences in the quantitative descriptors of corneal topography between corneas with keratoconus from unilateral and bilateral cases. The authors' results suggest that the incidence of "unilateral" keratoconus is very low. Patients initially diagnosed with unilateral keratoconus, if observed for a sufficient period, commonly had signs of keratoconus develop in the opposite eye. The possibility that all cases of unilateral keratoconus may eventually become bilateral cannot be excluded. Therefore, refractive surgical procedures should not be performed on apparently normal corneas when keratoconus is detected in the opposite eye.

Effect of ongoing treatment of amblyopia on surgical outcome in esotropia.

PURPOSE: Classic teaching recommends completion of amblyopia therapy prior to surgical correction of esotropia. Recent reports, however, suggest that incomplete treatment does not adversely affect surgical outcome. This study assesses the effect of incompletely treated amblyopia on the success rate of bimedial rectus recession in infantile and acquired esotropia. METHODS: All patients (n = 102) with esotropia undergoing bimedial rectus recession in 1994 who met inclusion criteria were reviewed. Subjects were classified as having infantile; acquired, partially accommodative; or acquired, nonaccommodative esotropia for comparison. Amblyopia was classified as none, mild, moderate, or severe. Surgical success was defined as orthophoria +/- 8 prism diopters and was assessed at the second postoperative visit (4 to 6 weeks after surgery). Other variables studied included mean surgical age, preoperative deviation, millimeters of surgery, and amount of follow up. RESULTS: For all patients, surgical success rates were as follows: no amblyopia, 84.3% (43/51); mild amblyopia, 81.6% (31/38); and moderate amblyopia, 61.5% (8/13). All patients with severe amblyopia underwent unilateral recess/resect procedures and were excluded. Of the esotropia subgroups, a statistically significant decrease in surgical success was noted only in the infantile esotropia group with moderate amblyopia. For this group, success rates were as follows: no amblyopia, 77.1% (27/35); mild amblyopia, 81.0% (17/21); and moderate amblyopia, 16.7% (1/6), P = 0.005. CONCLUSIONS: Performing corrective surgery on patients with infantile esotropia leads to poorer surgical outcome if moderate amblyopia is present at the time of surgery. Mild amblyopia, however, does not adversely affect surgical outcome in patients with infantile esotropia. Furthermore, the presence of mild or moderate amblyopia does not appear to have an influence on surgical outcome for patients with acquired esotropia. The effect of amblyopia on sensory outcome was not studied as most patients were too young for reliable sensory testing.

Structural analysis of zinc substitutions in the active site of thermolysin.

Native thermolysin binds a single catalytically essential zinc ion that is tetrahedrally coordinated by three protein ligands and a water molecule. During catalysis the zinc ligation is thought to change from fourfold to fivefold. Substitution of the active-site zinc with Cd2+, Mn2+, Fe2+, and Co2+ alters the catalytic activity (Holmquist B, Vallee BL, 1974, J Biol Chem 249:4601-4607). Excess zinc inhibits the enzyme. To investigate the structural basis of these changes in activity, we have determined the structures of a series of metal-substituted thermolysins at 1.7-1.9 A resolution. The structure of the Co(2+)-substituted enzyme is shown to be very similar to that of wild type except that two solvent molecules are liganded to the metal at positions that are thought to be occupied by the two oxygens of the hydrated scissile peptide in the transition state. Thus, the enhanced activity toward some substrates of the cobalt-relative to the zinc-substituted enzyme may be due to enhanced stabilization of the transition state. The ability of Zn2+ and Co2+ to accept tetrahedral coordination in the Michaelis complex, as well as fivefold coordination in the transition state, may also contribute to their effectiveness in catalysis. The Cd(2+)- and Mn(2+)-substituted thermolysins display conformational changes that disrupt the active site to varying degrees and could explain the associated reduction of activity. The conformational changes involve not only the essential catalytic residue, Glu 143, but also concerted side-chain rotations in the adjacent residues Met 120 and Leu 144. Some of these side-chain movements are similar to adjustments that have been observed previously in association with the "hinge-bending" motion that is presumed to occur during catalysis by the zinc endoproteases. In the presence of excess zinc, a second zinc ion is observed to bind at His 231 within 3.2 A of the zinc bound to native thermolysin, explaining the inhibitory effect.

Thermal stability determinants of chicken egg-white lysozyme core mutants: hydrophobicity, packing volume, and conserved buried water molecules.

A series of 24 mutants was made in the buried core of chicken lysozyme at positions 40, 55, and 91. The midpoint temperature of thermal denaturation transition (Tm) values of these core constructs range from 60.9 to 77.3 degrees C, extending an earlier, more limited investigation on thermostability. The Tm values of variants containing conservative replacements for the wild type (WT) (Thr 40-Ile 55-Ser 91) triplet are linearly correlated with hydrophobicity (r = 0.81) and, to a lesser degree, with combined side-chain volume (r = 0.75). The X-ray structures of the S91A (1.9 A) and I55L/S91T/D101S (1.7 A) mutants are presented. The former amino acid change is found in duck and mammalian lysozymes, and the latter contains the most thermostable core triplet. A network of four conserved, buried water molecules is associated with the core. It is postulated that these water molecules significantly influence the mutational tolerance at the individual triplet positions. The pH dependence of Tm for the S91D mutant was compared with that of WT enzyme. The pKa of S91D is 1.2 units higher in the native than in the denatured state, corresponding to delta delta G298 = 1.7 kcal/mol. This is a low value for charge burial and likely reflects the moderating influence of the buried water molecules or a conformational change. Thermal and chemical denaturation and far UV CD spectroscopy were used to characterize the in vitro properties of I55T. This variant, which buries a hydroxyl group, has similar properties to those of the human amyloidogenic variant I56T.

Nerve growth factor in different crystal forms displays structural flexibility and reveals zinc binding sites.

Murine beta-nerve growth factor (beta NGF) is a 118 amino acid residue polypeptide which, as a functional dimer, plays an important role in the survival and development of certain neuronal populations. The structure of the bis-desocta1-8 form of murine beta NGF has been determined in two different crystal modifications using X-ray methods. The two crystal forms, with space groups P2(1)2(1)2(1) and C2, were grown from 18 to 20% polyethylene glycol 8000 and 100 mM Pipes (pH 6.1) with zinc acetate concentrations of 1 mM and 100 mM, respectively. The C2 structure was solved by multiple isomorphous replacement using four heavy-atom derivatives and was refined to a crystallographic residual of 17.9% and 2.5 A resolution. The crystals contain three beta NGF monomers per asymmetric unit. Two monomers form a dimer related by a non-crystallographic 2-fold axis of symmetry. The third monomer also forms a dimer that is very similar, but with a crystallography related monomer as a partner. The electron density clearly defines residues 12 through 115 for all three monomers but the extreme N and C-terminal residues (9 to 11, 116 to 118) are ill defined in some cases. The P2(1)2(1)2(1) structure was solved by molecular replacement using the C2 structure as a search model and was refined to a crystallographic residual of 19.7% at 2.8 A resolution. This crystal form contains two monomers per asymmetric unit, again arranged as a non-crystallographic 2-fold-related dimer. The N and c termini are also variably defined. The core of each of the five monomers, which forms a cysteine knot motif, is very similar in all structures. Also, the dimer structures are very similar to one another, whether the monomers are related by crystallographic or non-crystallographic symmetry. However, three of the four loop regions that extend from the core of each monomer display substantial variability in conformation, even between monomers of the same dimer. This structural variability in the putative receptor binding regions suggests that structural malleability might be important in allowing the ligands to bind to different receptors with different affinities.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparative QSAR studies of two series of 1,4-dihydropyridines as slow calcium channel blockers.

Quantitative structure activity analysis was applied to two series of dihydropyridine (DHP) calcium channel blocking agents. One series of compounds was composed of DHPs substituted in the 4-position with an ortho or meta nitro substituted phenyl ring. The second group consisted of DHPs substituted at the 4-position with a novel thieno [3,2-c] pyridine ring. Both series consisted of compounds with unsymmetrical ester substitutions on the dihydropyridine ring. The antihypertensive activity of the compounds were determined in a spontaneously hypertensive rat model. Regression analysis indicated the antihypertensive activity of an i.v. dose correlated with the calculated octanol/water coefficent (clogP). Regression analysis did not find correlation with the in vitro potency and the clogP values.

Inhibition of thermolysin and neutral endopeptidase 24.11 by a novel glutaramide derivative: X-ray structure determination of the thermolysin-inhibitor complex.

Determination of the X-ray structure of thermolysin-inhibitor complexes has proven useful in aiding our understanding of the mode of binding of inhibitors of related, physiologically important, mammalian zinc peptidases including neutral endopeptidase EC 3.4.24.11 and angiotensin-converting enzyme. Here we describe the mode of binding to crystalline thermolysin of N-[1-(2(R,S)-carboxy-4-phenylbutyl)-cyclopentylcarbonyl]-(S) -tryptophan (CCT). CCT is an analogue of both candoxatrilat, a potent inhibitor of neutral endopeptidase 24.11, and of the 5-indanyl ester prodrug candoxatril, which is under clinical evaluation as a potential therapy for congestive heart failure. CCT differs from the previously studied N-carboxyalkyl dipeptide CLT [N-(S)-(1-carboxy-3-phenylpropyl)-(S)-leucyl-(S)-tryptophan] in several important respects. It has a highly constrained gem-cyclopentyl P1' substituent and lacks the characteristic imino nitrogen substituent of CLT. The structure determination shows that, notwithstanding the conformational influence of the gem-cyclopentyl substituent, CCT binds within the active site of thermolysin in a similar manner to CLT. Although the characteristic hydrogen bond between the imino nitrogen of CLT and thermolysin is absent in CCT, the affinities of the two inhibitors for the enzyme are virtually identical. These results illustrate the importance of considering not only those hydrogen bonds that are formed in an enzyme-ligand complex but also the other hydrogen bonds that may be lost due to desolvation of the enzyme and ligand on formation of the complex. In addition, the overall conformational demands placed upon a ligand in order to achieve receptor interaction may be critically important.

Structural comparison suggests that thermolysin and related neutral proteases undergo hinge-bending motion during catalysis.

Crystal structures are known for three members of the bacterial neutral protease family: thermolysin from Bacillus thermoproteolyticus (TLN), the neutral protease from Bacillus cereus (NEU), and the elastase of Pseudomonas aeruginosa (PAE), both in free and ligand-bound forms. Each enzyme consists of an N-terminal and C-terminal domain with the active site formed at the junction of the two domains. Comparison of the different molecules reveals that the structure within each domain is well conserved, but there are substantial hinge-bending displacements (up to 16 degrees) of one domain relative to the other. These domain motions can be correlated with the presence or absence of bound inhibitor, as was previously observed in the specific example of PAE [Thayer, M.M., Flaherty, K.M., & McKay, D.B. (1991) J. Biol. Chem. 266, 2864-2871]. The binding of inhibitor appears to be associated with a reduction of the domain hinge-bending angle by 6-14 degrees and a closure of the "jaws" of the active site cleft by about 2 A. Crystallographic refinement of the structure of thermolysin suggests that electron density seen in the active site of the enzyme in the original structure determination probably corresponds to a bound dipeptide. Thus, the crystal structure appears to correspond to an enzyme-inhibitor or enzyme-product complex, rather than the free enzyme, as has previously been assumed.

Crystallization of the NAD-dependent malic enzyme from the parasitic nematode Ascaris suum.

The malic enzyme from muscle mitochondria of the parasitic nematode Ascaris suum is a tetramer of 65 kDa monomers that catalyzes the oxidative decarboxylation of malate to pyruvate and CO2 with NAD cofactor as oxidant. This malic enzyme is critical to the nematode for muscle function under anaerobic conditions. Unlike mammalian versions of the enzyme such as that found in rat liver, which require NADP as cofactor, the nematode version is an NAD-dependent enzyme. We report the crystallization of samples of the nematode enzyme at room temperature from pH 7.5 solutions of polyethylene glycol 4000 containing magnesium sulfate, NAD and sodium tartronate. Immediately upon mixing of protein and precipitant solutions, a marked precipitation of the protein occurs. Out of this precipitate, crystals appear almost immediately, most commonly in a truncated cube form that can grow to 0.5 to 0.7 mm on a cube edge in two to three days. The crystals are trigonal, space group P3(1)21 or its enantiomer, with a = b = 131.2(7) A, c = 152.6(9) A, and two monomers per asymmetric unit. Fresh crystals diffract X-radiation from a synchrotron source (lambda = 0.95 A) to about 3.0 A resolution. Rotational analysis of Patterson functions indicates that the malic enzyme tetramer has 222 symmetry.

The crystal structure of a lysine 49 phospholipase A2 from the venom of the cottonmouth snake at 2.0-A resolution.

The crystal structure of a lysine 49 variant phospholipase A2 (K49 PLA2) has been determined at 2.0-A resolution. This particular phospholipase A2, purified from the venom of the eastern cottonmouth (Agkistrodon piscivorus piscivorus), a North American pit viper, differs significantly from others studied crystallographically because of replacement of the aspartate residue at position 49, whose side chain is important in calcium binding, by lysine. The crystallographic analysis of K49 PLA2 was undertaken to assess the structural ramifications of this substitution, particularly as they affect the binding mechanism of both the calcium cofactor and the phospholipid substrate. The protein crystals are tetragonal, space group P4(1)2(1)2, with unit cell dimensions of a = b = 71.7 (1) and c = 57.8 (3) A. Preliminary phases were obtained by molecular replacement techniques with a search model derived from the refined 2.5-A structure of a rattle-snake venom phospholipase A2 (Brunie, S., Bolin, J., Gewirth, D., and Sigler, P. B. (1985) J. Biol. Chem. 260, 9742-9749). The starting model gave an initial crystallographic RF of 0.526 (RF = sigma parallel to Fo /-/ Fc parallel to /sigma/Fo/). The structure was refined against all data to 2.0-A resolution. The final RF is 0.158. The final model includes 150 discrete water molecules. The K49 PLA2 model is composed primarily of alpha-helices joined by loops, some of which are quite extensive. Although dissimilarities are observed in the loop regions, the helical portions are very similar to those in other known phospholipase A2 structures. The proposed catalytic center (His48, Tyr73, and Asp99) is also structurally conserved. The region in K49 PLA2 corresponding to the calcium-binding site in other phospholipases A2 is occupied by the epsilon-amino group of lysine 49.

Crystal structure of recombinant human interleukin-1 beta at 2.0 A resolution.

The crystal structure of recombinant human interleukin-1 beta (IL-1 beta) has been determined at 2.0 A resolution and refined to a crystallographic R-factor of 0.19. Three heavy-atom derivatives were identified and used for multiple isomorphous replacement phasing. Interpretation of the resulting electron density map revealed a structure in which there are 12 antiparallel beta-strands and no alpha-helix. The single 153-residue polypeptide chain is folded into a six-stranded beta-barrel similar in architecture to the Kunitz-type trypsin inhibitor found in soybeans. The molecule displays approximate 3-fold symmetry about the axis of the beta-barrel. Each successive pair of component strands of the barrel brackets an extensive sequence outside the barrel that includes an additional pair of beta-strands and a prominent loop. Together, these three external segments conceal much of the perimeter and one end of the barrel, leaving only the end supporting the chain termini fully exposed. The structure can be used to identify portions of the polypeptide chain that are exposed on the surface of the molecule, some of which must be epitopes recognized by interleukin-1 beta receptors.