ATP-dependent

No abstract

The topological similarity of IL-10 to IFN gamma was totally unexpected, and may be a reflection of the close relationship between the biological effects of these two cytokines. The structure of IL-10 provides insights into the possible modes of conversion of the dimer into monomers, and of putative sites of receptor interactions. The good level of refinement and high resolution of this structure show that the internal disorder often associated with other helical cytokines is not an essential feature of this class of proteins.

The crystal structure of a 1:1 complex between the German cockroach allergen Bla g 2 and the Fab fragment of a monoclonal antibody 7C11 was solved at 2.8-Å resolution. Bla g 2 binds to the antibody through four loops that include residues 60 -70, 83-86, 98 -100, and 129 -132. Cation-interactions exist between Lys-65, Arg-83, and Lys-132 in Bla g 2 and several tyrosines in 7C11. In the complex with Fab, Bla g 2 forms a dimer, which is stabilized by a quasi-four-helix bundle comprised of an ␣-helix and a helical turn from each allergen monomer, exhibiting a novel dimerization mode for an aspartic protease. A disulfide bridge between C51a and C113, unique to the aspartic protease family, connects the two helical elements within each Bla g 2 monomer, thus facilitating formation of the bundle. Mutation of these cysteines, as well as the residues Asn-52, Gln-110, and Ile-114, involved in hydrophobic interactions within the bundle, resulted in a protein that did not dimerize. The mutant proteins induced less ␤-hexosaminidase release from mast cells than the wild-type Bla g 2, suggesting a functional role of dimerization in allergenicity. Because 7C11 shares a binding epitope with IgE, the information gained by analysis of the crystal structure of its complex provided guidance for site-directed mutagenesis of the allergen epitope. We have now identified key residues involved in IgE antibody binding; this information will be useful for the design of vaccines for immunotherapy.Cockroach allergy is associated with the development of asthma and is a risk factor for emergency room admission of asthmatic patients, especially among inner city children living in low-income houses infested with cockroaches (1, 2). Cockroaches release allergens to the environment, which are carried by particles (5-40 m of diameter) that reach the lung by inhalation. Bla g 2 is one of the most important cockroach allergens, eliciting production of specific IgE in ϳ70% of cockroach-allergic patients at exposure levels that are 10 -100-fold lower than those from other common indoor allergens from dust mite and cat (3-5). Exposure to Bla g 2 results in cross-linking of IgE bound to the surface of mast cells or basophils from sensitized patients (i.e. in immunological terms, cross-linking refers to the non-covalent linkages between the allergen and two IgE molecules at the surface of mast cells or basophils), and induces release of potent mediators (histamine, leukotrienes, prostaglandins, etc.) of allergic reactions.We recently solved the crystal structure of Bla g 2, confirming that the overall fold of this allergen corresponds to that of pepsin-like aspartic proteases, and revealing structural elements that explain why it is enzymatically inactive (6, 7). Bla g 2 contains important amino acid substitutions in the area corresponding to the catalytic site. These modifications impair enzymatic function, and the allergen did not show proteolytic activity in standard in vitro assays using casein and hemoglobin as substrates (7). Bla g 2 belongs to a gro...

The structures of recombinant histo-aspartic protease (HAP) from malaria-causing parasite Plasmodium falciparum, as apoenzyme and in complex with two inhibitors, pepstatin A and KNI-10006, were solved at 2.5, 3.3, and 3.05 Å resolution, respectively. In the apoenzyme crystals HAP forms a tight dimer, not seen previously in any aspartic proteases. The interactions between the monomers affect the conformation of two flexible loops, the functionally important "flap" (residues 70-83) and its structural equivalent in the C-terminal domain (238-245), as well as the orientation of the helix 225-235. The flap is found in an open conformation in the apoenzyme. Unexpectedly, the active site of the apoenzyme contains a zinc ion tightly bound to His32 and Asp215 from one monomer, and to Glu278A from the other monomer, with the coordination of Zn resembling that seen in metalloproteases. The flap is closed in the structure of the pepstatin A complex, whereas it is open in the complex with KNI-10006. Although the binding mode of pepstatin A is significantly different than in other pepsin-like aspartic proteases, its location in the active site makes unlikely the previously proposed hypothesis that HAP is a serine protease. The binding mode of KNI-10006 is unusual compared to the binding of other inhibitors from the KNI series to aspartic proteases. The novel features of the HAP active site could facilitate design of specific inhibitors used in the development of antimalarial drugs.

The crystal structure of a recombinant form of the proteinase encoded by the feline immunodeficiency virus (FIV PR) has been solved at 2 A resolution and refined to an R-factor of 0.148. The refined structure includes a peptidomimetic, statine-based inhibitor, LP-149, which is an even more potent inhibitor of HIV PR. Kinetic parameters were obtained for the cleavage of five substrates by FIV PR, and inhibition constants were measured for four inhibitors. The structure of FIV PR resembles other related retroviral enzymes although few inhibitors of HIV PR are capable of inhibiting FIV PR. The structure of FIV PR will enhance our knowledge of this class of enzymes, and will direct testing of new proteinase inhibitors in a feline animal model.

The yeast IA 3 polypeptide consists of only 68 residues, and the free inhibitor has little intrinsic secondary structure. IA 3 showed subnanomolar potency toward its target, proteinase A from Saccharomyces cerevisiae, and did not inhibit any of a large number of aspartic proteinases with similar sequences/structures from a wide variety of other species. Systematic truncation and mutagenesis of the IA 3 polypeptide revealed that the inhibitory activity is located in the N-terminal half of the sequence. Crystal structures of different forms of IA 3 complexed with proteinase A showed that residues in the N-terminal half of the IA 3 sequence became ordered and formed an almost perfect ␣-helix in the active site of the enzyme. This potent, specific interaction was directed primarily by hydrophobic interactions made by three key features in the inhibitory sequence. Whereas IA 3 was cut as a substrate by the nontarget aspartic proteinases, it was not cleaved by proteinase A. The random coil IA 3 polypeptide escapes cleavage by being stabilized in a helical conformation upon interaction with the active site of proteinase A. This results, paradoxically, in potent selective inhibition of the target enzyme.Aspartic proteinases participate in a variety of physiological processes, and the onset of pathological conditions such as hypertension, gastric ulcers, and neoplastic diseases may be related to changes in the levels of their activity. Members of this proteinase family, e.g. renin, pepsin, cathepsin D, and human immunodeficiency virus-proteinase are generally typecast on the basis of their susceptibility to inhibition by naturally occurring, small molecule inhibitors such as the acylated pentapeptides, isovaleryl-and acetyl-pepstatin. However, the two most recently identified human aspartic proteinases, ␤-site Alzheimer's precursor protein cleavage enzyme and ␤-site Alzheimer's precursor protein cleavage enzyme 2 (1, 2), are not inhibited by this classical type of inhibitor of this family of enzymes. Pepstatins are metabolic products produced by various species of actinomycetes and, as such, are not themselves gene-encoded. Protein inhibitors of aspartic proteinases are relatively uncommon and are found in only a few specialized locations (3). Examples include renin-binding protein in mammalian kidneys which intriguingly has now itself been identified to be the enzyme, N-acetyl-D-glucosamine-2-epimerase (4); a 17-kDa inhibitor of pepsin and cathepsin E from the parasite, Ascaris lumbricoides (5); proteins from plants such as potato, tomato, and squash (6, 7), and a pluripotent inhibitor from sea anemone of cysteine proteinases as well as cathepsin D (8).The IA 3 polypeptide in yeast is an 8-kDa inhibitor of the vacuolar aspartic proteinase (proteinase A or saccharopepsin) that was initially described by Holzer and co-workers (9). The complete sequence of this 68-residue inhibitor has been elucidated (10, 11) and the inhibitory activity of IA 3 has been shown to reside within the N-terminal half of the molecule (10, 12). ...

The different isolates available for HIV-1 and HIV-2 were compared for the region of the protease (PR) sequence, and the variations in amino acids were analyzed with respect to the crystal structure of HIV-1 PR with inhibitor. Based on the extensive homology (39 identical out of 99 residues), models were built of the HIV-2 PR complexed with two different aspartic protease inhibitors, acetylpepstatin and a renin inhibitor, H-261. Comparison of the HIV-1 PR crystal structure and the HIV-2 PR model structure and the analysis of the changes found in different isolates showed that correlated substitutions occur in the hydrophobic interior of the molecule and at surface residues involved in ionic or hydrogen bond interactions. The substrate binding residues of HIV-1 and HIV-2 PRs show conservative substitutions of four residues. The difference in affinity of HIV-1 and HIV-2 PRs for the two inhibitors appears to be due in part to the change of Val 32 in HIV-1 PR to Ile in HIV-2 PR.