Identification of the high affinity binding site in transforming growth factor-beta involved in complex formation with alpha 2-macroglobulin. Implications regarding the molecular mechanisms of complex formation between alpha 2-macroglobulin and growth factors, cytokines, and hormones.

The biological activities of transforming growth factor-beta isoforms (TGF-beta(1,2)) are known to be modulated by alpha(2)-macroglobulin (alpha(2)M). alpha(2)M forms complexes with numerous growth factors, cytokines, and hormones, including TGF-beta. Identification of the binding sites in TGF-beta isoforms responsible for high affinity interaction with alpha(2)M many unravel the molecular basis of the complex formation. Here we demonstrate that among nine synthetic pentacosapeptides with overlapping amino acid sequences spanning the entire TGF-beta(1) molecule, the peptide (residues 41-65) containing Trp-52 exhibited the most potent activity in inhibiting the formation of complexes between (125)I-TGF-beta(1) and activated alpha(2)M (alpha(2)M*) as determined by nondenaturing polyacrylamide gel electrophoresis and by plasma clearance in mice. TGF-beta(2) peptide containing the homologous sequence and Trp-52 was as active as the TGF-beta(1) peptide, whereas the corresponding TGF-beta(3) peptide lacking Trp-52, was inactive. The replacement of the Trp-52 with alanine abolished the inhibitory activities of these peptides. (125)I-TGF-beta(3), which lacks Trp-52, bound to alpha(2)M* with an affinity lower than that of (125)I-TGF-beta(1). Furthermore, unlabeled TGF-beta(3) and the mutant TGF-beta(1)W52A, in which Trp-52 was replaced with alanine, were less potent than unlabeled TGF-beta(1) in blocking I(125)-TGF-beta(1) binding to alpha(2)M*. TGF-beta(1) and TGF-beta(2) peptides containing Trp-52 were also effective in inhibiting I(125)-nerve growth factor binding to alpha(2)M*. Tauhese results suggest that Trp-52 is involved in high affinity binding of TGF-beta to alpha(2)M*. They also imply that TGF-beta and other growth factors/cytokines/hormones may form complexes with alpha(2)M* via a common mechanism involving the interactions between topologically exposed Trp and/or other hydrophobic residues and a hydrophobic region in alpha(2)M*.

Synthesis and activity of selective MMP inhibitors with an aryl backbone.

A series of novel, MMP-1 sparing arylhydroxamate sulfonamides with activity against MMP-2 and -13 is described.

An active site of transforming growth factor-beta(1) for growth inhibition and stimulation.

Transforming growth factor-beta (TGF-beta) is a bifunctional growth regulator. It inhibits growth of many cell types, including epithelial cells, but stimulates growth of others (e.g. fibroblasts). The active site on the TGF-beta molecule, which mediates its growth regulatory activity, has not been defined. Here, we show that antibody to a TGF-beta(1) peptide containing the motif WSLD (52nd to 55th amino acid residues) completely blocked both (125)I-TGF-beta(1) binding to TGF-beta receptors and TGF-beta(1)-induced growth inhibition in mink lung epithelial cells. Site-directed mutagenesis analysis revealed that the replacement of Trp(52) and Asp(55) by alanine residues diminished the growth inhibitory activity of TGF-beta(1) by approximately 90%. Finally, while wild-type TGF-beta(1) was able to stimulate growth of transfected NIH 3T3 cells, the double mutant TGF-beta(1) W52A/D55A was much less active. These results support the hypothesis that the WSLD motif is an active site of TGF-beta(1), which is important for growth inhibition of epithelial cells and growth stimulation of fibroblasts.

Synthesis and identification of conformationally constrained selective MMP inhibitors.

We have discovered a new series of potent conformationally constrained MMP Inhibitors that are selective for MMP-13 over MMP-1.

Three-dimensional structure of human cytomegalovirus protease.

Herpesviruses encode a serine protease that specifically cleaves assembly protein. This protease is critical for replication, and represents a new target for antiviral drug design. Here we report the three-dimensional structure of the protease from human cytomegalovirus (hCMV) at 2.27 angstroms resolution. The structure reveals a unique fold and new catalytic strategy for cleavage. The monomer fold of the enzyme, a seven-stranded beta-barrel encircled by a chain of helices that form the carboxy terminus of the molecule, is unrelated to those observed in classic serine proteases such as chymotrypsin and subtilisin. The serine nucleophile at position 132 is activated by two juxtaposed histidine residues at positions 63 and 157. Dimerization, which seems to be necessary for activity, is observed in the crystals. Correlations of the structure with the sequences of herpesvirus proteases suggest that dimerization may confer specificity and recognition in substrate binding.

Using sampling techniques in protein crystallization.

The crystallization of homogeneous or highly purified macromolecules depends on many variables such as precipitant, pH, choice of buffer, protein concentration, temperature, the participation of different mono- and divalent ions, as well as the presence of minute amounts of detergent and organic molecules. Finding the best combination among these many parameters is a multi-variable optimization problem. This kind of problem can be treated mathematically by sampling techniques. We have used this technique for protein crystallization. The iterative procedure starts with random sampling, followed by quantitative evaluation and cycles with weighted sampling. A simple procedure, derived from this concept and called MON48, has been successfully applied to many protein crystallization problems.

Renin inhibitor SC-51106 complexed with human renin: discovery of a new binding site adjacent to P3.

SC-51106, a 'minimal-size' diol-based renin inhibitor lacking a P4 residue, has been co-crystallized with human renin and the structure of the complex determined by X-ray crystallography. This study defines the mode of binding of this important class of renin inhibitor, and in conjunction with molecular modeling, has led to the discovery of a new binding site adjacent to S3, which is termed the 'S3aux(iliary)' subsite.

Structure and topological symmetry of the glyphosate target 5-enolpyruvylshikimate-3-phosphate synthase: a distinctive protein fold.

5-enol-Pyruvylshikimate-3-phosphate synthase (EPSP synthase; phosphoenolpyruvate:3-phosphoshikimate 1-carboxyvinyltransferase, EC 2.5.1.19) is an enzyme on the pathway toward the synthesis of aromatic amino acids in plants, fungi, and bacteria and is the target of the broad-spectrum herbicide glyphosate. The three-dimensional structure of the enzyme from Escherichia coli has been determined by crystallographic techniques. The polypeptide backbone chain was traced by examination of an electron density map calculated at 3-A resolution. The two-domain structure has a distinctive fold and appears to be formed by 6-fold replication of a protein folding unit comprising two parallel helices and a four-stranded sheet. Each domain is formed from three of these units, which are related by an approximate threefold symmetry axis; in each domain three of the helices are completely buried by a surface formed from the three beta-sheets and solvent-accessible faces of the other three helices. The domains are related by an approximate dyad, but in the present crystals the molecule does not display pseudo-symmetry related to the symmetry of point group 32 because its approximate threefold axes are almost normal. A possible relation between the three-dimensional structure of the protein and the linear sequence of its gene will be described. The topological threefold symmetry and orientation of each of the two observed globular domains may direct the binding of substrates and inhibitors by a helix macrodipole effect and implies that the active site is located near the interdomain crossover segments. The structure also suggests a rationale for the glyphosate tolerance conferred by sequence alterations.

Crystallization of a chymotrypsin inhibitor from Erythrina caffra seeds.

Crystals of a chymotrypsin inhibitor from Erythrina caffra seeds have been grown out of lithium sulfate, by the hanging drop method of vapor diffusion. The crystals belong to the rhombohedral space group R32, with a = 67.2 A and alpha = 99.4 degrees, and diffract to 3 A resolution.

Three-dimensional structure of a genetically engineered variant of porcine growth hormone.

The three-dimensional structure of a genetically engineered variant of porcine growth hormone, methionyl porcine somatotropin (MPS), has been determined at 2.8-A resolution, using single crystal x-ray diffraction techniques. Phases were obtained by use of a single isomorphous K2OsCl6 derivative and were improved by use of the density modification procedure. The MPS structure is predominantly helical. It consists mainly of four antiparallel alpha-helices arranged in a left twisted helical bundle, a structural motif observed in a number of other unrelated proteins. However, the way the four helices are connected in the bundle is unusual and, to our knowledge, has never been reported before. Alignment of the amino acid sequence of MPS with that of other growth hormones reveals that residues within the alpha-helices are predominantly invariant and thus these invariant residues are necessary to maintain the structural integrity of these proteins.

Molecular complex of lumiflavin and 2-aminobenzoic acid: crystal structure, crystal spectra, and solution properties.

The molecular complex lumiflavin-2-aminobenzoic acid monohydrate (C13H12N4O2.C7H7NO2.H2O) crystallizes from from aqueous solution as red triclinic prisms. The space group is P1 with cell dimensions a = 9.660 A, b = 14.866 A, c = 7.045 A, alpha = 95.44 degrees , beta = 95.86 degrees, and gamma = 105.66 degrees . The crystal structure was solved by direct methods and refined by block-diagonal least-squares procedures to an R value of 0.050 on the basis of 1338 observed reflections. The structure is composed of stacks of alternating lumiflavin adn un-ionized (neutral) 2-aminobenzoic acid molecules. Two different modes of stacking interaction are observed. In one, 2-aminobenzoic acid overlaps all three of the isoalloxazine rings, at a mean distance of 3.36 A; in the other, 2-aminobenzoic acid interacts distance of 3.36 A; in the other, 2-aminobenzoic acid interacts with the pyrazine and dimethylbenzene moieties, at a distance of 3.42 A. Perpendicular to the stacking direction, the molecules form a continuous sheet. Each flavin is hydrogen bonded via O(2) and NH(3) to two symmetrically related aminobenzoates; the water of crystallization forms three hydrogen bonds, bridging two flavins, via O(4) and N(5), and one aminobenzoic acid. The red color of the crystals results from a charge-transfer transition involving stacked flavin and 2-aminobenzoic acid. The red color of the crystals results from a charge-transfer transition involving stacked flavin and 2-aminobenzoic acid molecules. Measurements of the polarized optical absorption spectra of crystals show that the transition moment direction for the long wavelength absorbance (beyond 530 nm) contains an out-of-plane component which can only arise from a charge-transfer interaction. Since the amino N does not make exceptionally close interactions with isoalloxazine atoms in either stacking mode (minimum interatomic distance 3.52 A), the charge transfer is presumed to involve pi orbitals of the 2-aminobenzoic acid donor.

Highly structured water network in crystals of a deoxydinucleoside---drug complex.

X-ray diffraction analysis of crystals of the intercalative complex between the deoxyribonucleoside phosphate d(CpG) and the mutagen proflavine shows a highly structured arrangement of water molecules linked together by newtworks of hydrogen bonds to form four edge-linked pentagons per asymmetric unit. These pentagons have a general role in maximizing hydrogen bonding at 3.4-A intervals. The conformation of the deoxyribose sugar ring at the 3' end of one strand can depend on its local aqueous environment.

The structure of drug-deoxydinucleoside phosphate complex; generalized conformational behavior of intercalation complexes with RNA and DNA fragments.

A 2:2 complex of proflavine and deoxycytidylyl-3', 5'-guanosine has been crystallized and its structure determined by x-ray crystallography. The two dinucleoside phosphate strands form self complementary duplexes with Watson Crick hydrogen bonds. One proflavin is asymmetrically intercalated between the base pairs and the other is stacked above them. The conformations of the nucleotides are unusual in that one strand has C3',C2'endomixed sugar puckering and the other has C3',C3' endo deoxyribose sugars. These results show that the conformation of the 3'sugar is of secondary importance to the intercalated geometry.

Effect of arginine analogues on a fish pathogen, Aeromonas salmonicida.

The effects of arginine analogues on a fish pathogen, Aeromonas salmonicida, were investigated. This organism proved to be resistant in various degrees to most arginine analogues tested but was found to be sensitive to arginine hydroxamate. Inhibition of growth by arginine hydroxamate was reversed by the addition of arginine to the bacterial culture. Incorporation of 14C from 14C-amino acids, namely, guanidoarginine, glutamate, asparate, alanine, isoleucine, serine, methyl-methionine, and cystine was affected.

Effect of aeromonas salmonicida (furunculosis) infection on the incorporation of [1-14C]acetate into lipids of trout liver.

[1-(14)C]acetate is incorporated into lipids more rapidly by liver from Aeromonas salmonicida-infected trout than by normal trout liver, both in vitro and in vivo. The increase was, in general, about twofold to less than threefold.

Blood changes in brook trout induced by infection with Aeromonas salmonicida.

Furunculosis was induced in brook trout, Salvelinus fontinalis, by experimental inoculation with Aeromonas salmonicida. Total protein, hemoglobin, sialic acid, fatty acids, triglycerides, cholesterol, inorganic-phosphorus, acid-soluble phosphorus, and lipid-phosphorus decreased in the blood of the infected fish while amino acids, urea, total creatinine, ammonia, and glucose increased. Pyruvic acid, lactic acid, and ascorbic acid values showed no significant change.