[Forms of LonB protease from Archaeoglobus fulgidus devoid of the transmembrane domain: the contribution of the quaternary structure to the regulation of enzyme proteolytic activity].

Deletion of the transmembrane domain (TM-domain) of Archaeoglobus flggidus LonB protease (AfLon) was shown to result in uncontrollable activation of the enzyme proteolytic site and in vivo autolysis yielding a stable and functionally inactive fragment consisting of both alpha-helical and proteolytic domains (alphaP). The deltaTM-AfLonTM-S590A enzyme form, obtained by site-directed mutagenesis of the catalytic Ser residue, is capable of recombination with the alphaP fragment. The mixed oligomers were shown to be proteolytically active, which indicates a crucial role of subunit interactions in the activation of the AfLon proteolytic site. The thermophilic nature of AfLon protease was found to be due to the special features of the enzyme activity regulation, the structure of ATPase domain, and the quaternary structure.

Cyanovirin-N: a sugar-binding antiviral protein with a new twist.

Cyanovirin-N (CV-N), an 11-kDa protein from the cyanobacterium Nostoc ellipsosporum, is a highly potent virucidal agent that has generated interest as a lead natural product for the prevention and chemotherapy of human immunodeficiency virus infection. The antiviral activity of CV-N is mediated through specific, high-affinity interactions with the viral surface envelope glycoproteins. A number of structures of wild-type, mutant and sequence-shuffled CV-N have been solved by nuclear magnetic resonance and crystallography, showing that the protein exists as either a quasi-symmetric two-domain monomer or a domain-swapped dimer. Structures of several complexes of CV-N with oligosaccharides help in explaining the unique mode of high-affinity binding of these molecules to both forms of CV-N.

Crystal structure of a cyclic form of bovine pancreatic trypsin inhibitor.

The crystal structure of a cyclic form of a mutant of bovine pancreatic trypsin inhibitor has been solved at 1.0 A resolution. The protein was synthesized by native chemical ligation and its structure is almost indistinguishable from the previously described recombinant form of the same mutant; however, the new loop containing the former termini became much better ordered.

The structure of an insect chymotrypsin.

The South American imported fire ant (Solenopsis invicta), without natural enemies in the United States, widely infests the southern United States, causing more than a half billion dollars in health and agriculture-related damage annually in Texas alone. Fire ants are resistant to most insecticides, so control will require a more fundamental understanding of their biochemistry and metabolism leading to the design of selective, ecologically safe insecticides. The 4th instar larvae play a crucial role in the nutrition of the colony by secreting proteinases (especially chymotrypsin) which digest food products for the entire colony. The first structure of an ant proteolytic enzyme, fire ant chymotrypsin, was determined to atomic resolution (1.7 A). A structural comparison of the ant and mammalian structures confirms the "universality" of the serine proteinase motif and reveals a difference at residues 147-148, which are proteolytically removed in the bovine enzyme but are firmly intact in the ant chymotrypsin, suggesting a different activation mechanism for the latter. Likewise, the absence of the covalently attached propeptide domain (1-15) further suggests an uncharacteristic activation mechanism. The presence of Gly189 in the S1 site is an atypical feature of this chymotrypsin and is comparable only to human leukocyte elastase, hornet chymotrypsin and fiddler crab collagenase. Binding studies confirm the chymotrypsin nature of this novel enzyme.

Structure of recombinant mouse collagenase-3 (MMP-13).

The matrix metalloproteinases are crucial in the physiological and pathological degradation of the mammalian extracellular matrix, including breast tumours, and osteoarthritic cartilage. These enzymes are classified according to their matrix substrate specificity. Collagenase-3 (MMP-13) is a member of this family and preferentially cleaves type II collagen, cartilage, fibronectin and aggrecan. Collagenase-3 is normally expressed in hypertrophic chondrocytes, periosteal cells, and osteoblasts during bone development. The structure of the catalytic domain of recombinant mouse collagenase-3, complexed to the hydroxamate inhibitor (RS-113456), is reported at 2.0 A resolution. Molecular replacement and weak phasing information from a single derivative determined the structure. Neither molecular replacement nor derivative methods had a sufficient radius of convergence to yield a refinable structure. The structure illuminates the atomic zinc ion interactions with functional groups in the active site, emphasizing zinc ligation and the very voluminous hydrophobic P1' group for the inhibitor potency. The structure provides insight into the specificity of this enzyme, facilitating design of specific inhibitors to target various diseases.

Batimastat, a potent matrix mealloproteinase inhibitor, exhibits an unexpected mode of binding.

Matrix metalloproteinase enzymes have been implicated in degenerative processes like tumor cell invasion, metastasis, and arthritis. Specific metalloproteinase inhibitors have been used to block tumor cell proliferation. We have examined the interaction of batimastat (BB-94) with a metalloproteinase [atrolysin C (Ht-d), EC 3.4.24.42] active site at 2.0-angstroms resolution (R = 16.8%). The title structure exhibits an unexpected binding geometry, with the thiophene ring deeply inserted into the primary specificity site. This unprecedented binding geometry dramatizes the significance of the cavernous primary specificity site, pointing the way for the design of a new generation of potential antitumor drugs.

Structure-based analysis of inhibitor binding to Ht-d.

A theoretical study was performed on the structure of both the native and inhibited metalloproteinase Ht-d (E.C. 3.4.24.42) solved at 2.0 A resolution. The energy maps calculated by program GRID clearly showed the extended binding site of Ht-d and allowed localization and characterization of the pockets S1-S3 and S1'-S3'. The GRID energy contour maps point out the particular shape of the S1' pocket in agreement with experimental density maps and inhibited Ht-d structures. Based on the high degree of sequence homology of the Ht-d active site to that of mammalian metalloproteinases, the characterization of active site pockets was extended to neutrophil collagenase, fibroblast collagenase, stromelysin 1 and 2. Thirty residues of the Ht-d propeptide were modeled and optimized with reference to the Ht-d structure, giving insight to the mechanism of natural inhibition in metalloproteinase proenzymes. Kinetic measurements of Ht-d inhibition by a series of synthetic peptides show, in agreement with our Ht-d propeptide model, the crucial role of cysteine and adjacent residues in the specificity of Ht-d propeptide. This study suggests the structural link between Ht-d and mammalian metalloproteinases, contributing to the understanding of the mechanism of natural and synthetic inhibitor binding to metalloproteinases. Therefore, Ht-d is a good model system for the design of novel inhibitors against these enzymes with enhanced potency and specificity.

Structural interaction of natural and synthetic inhibitors with the venom metalloproteinase, atrolysin C (form d).

The structure of the metalloproteinase and hemorrhagic toxin atrolysin C form d (EC 3.4.24.42), from the venom of the western diamondback rattlesnake Crotalus atrox, has been determined to atomic resolution by x-ray crystallographic methods. This study illuminates the nature of inhibitor binding with natural (< Glu-Asn-Trp, where < Glu is pyroglutamic acid) and synthetic (SCH 47890) ligands. The primary specificity pocket is exceptionally deep; the nature of inhibitor and productive substrate binding is discussed. Insights gained from the study of these complexes facilitate the design of potential drugs to treat diseases where matrix metalloproteinases have been implicated, e.g., arthritis and tumor metastasis.

Iron absorption and loss in psoriasis.

59Fe absorption has been studied in psoriatics to elucidate the iron deficiency state. To determine the rate of iron loss, elimination of injected 59Fe was measured. In psoriasis mean iron absorption did not differ from the mean in the normal group, but a pathologically low absorption was found in 8 cases. Iron loss was significantly higher in psoriatics than in normal men, while it did not differ significantly from iron loss in women with regular menses.