Cathepsin D (EC 3.4.23.5) is a lysosomal protease suspected to play important roles in protein catabolism, antigen processing, degenerative diseases, and breast cancer progresson. Determination of the crystal structures of cathepsin D and a complex with pepstatin at 2.5 A resolution provides insights into inhibitor binding and lysosomal targeting for this two-chain, N-glycosylated aspartic protease. Comparison with the structures of a complex of pepstatin bound to rhizopuspepsin and with a human renin-bihbitor complex revealed differences in subsite structures and inhibitor-enzyme interactions that are consistent with affnity differences and structure-activity relationships and suggest strategies for fmetuning the specificity of cathepsin D inhibitors. Mutagenesis studies have identified a phosphotransferase recognition region that is required for oligosaccharide phosphorylation but is 32 A distant from the N-domain glycosylation site at Asn-70. Electron density for the crystal structure of cathepsin D indicated the presence of an N-linked oligosaccharide that extends from Asn-70 toward Lys-203, which is a key component of the phosphotransferase recognition region, and thus provides a structural explanation for how the phosphotransferase can recognize apparently distnt sites on the protein surface.Cathepsin D (EC 3.4.23.5) is an aspartic protease that is normally found in the lysosomes of higher eukaryotes where it functions in protein catabolism (1). This enzyme is distinguished from other members of the pepsin family (2) by two features that are characteristic of lysosomal hydrolases. First, mature cathepsin D is found predominantly in a twochain form due to a posttranslational cleavage event (1, 3). Second, it contains phosphorylated, N-linked oligosaccharides that target the enzyme to lysosomes via mannose 6-phosphate (M6P) receptors (4, 5). Phosphorylation involves recognition of both sugar and protein structural determinants by a phosphotransferase enzyme (6, 7).Interest in cathepsin D as a target for drug design results from its association with several biological processes of therapeutic significance including lysosomal biogenesis and protein targeting (4,5), antigen processing and the presentation of peptide fragments to class II major histocompatibility complexes (8-10), connective tissue disease pathology (11), muscular dystrophy (12), degenerative brain changes (13,14), and cleavage of amyloid precursor protein within senile plaques of Alzheimer brain (15). Recent duced in the vicinity of the growing tumor, may degrade the extracellular matrix and thereby promote the escape of cancer cells to the lymphatic and circulatory systems and enhance the invasion of new tissues (17,18). The design of potent and specific inhibitors of cathepsin D will aid the further elucidation of the roles of this enzyme in human disease. We previously described the purification and crystallization ofhuman cathepsin D from liver (3); similar studies have been reported recently for cathepsin D isolated from bovine l...

The incorporation of C2 symmetry has become a useful paradigm in the design of active site inhibitors for HIV-I protease (HIV PR) and has led to the design of a series of highly potent, C2 symmetry-based, diol-containing inhibitors of HIV PR, one of which, A-77003, has reached clinical trials. However, the stereochemistry of the diol core influences protease inhibition and antiviral activity in a manner that is not well understood. We analyzed the crystal structures of a diastereomeric series of C2 symmetry-based diol inhibitors, along with a deshydroxy analogue, bound to HIV PR and found that the stereochemistry of the diol core influences the mode of binding to the active site aspartic acids. Diasteromers with similar binding affinity can bind in different, asymmetric and symmetric, modes, while those with different binding affinities can bind in a similar manner. The positional symmetry of an inhibitor with respect to the enzyme C2 axis may be distinguished from its conformational symmetry. The structural differences between the inhibitor complexes were mainly confined to the central core portion of the diols, can be described by torsional differences about the central three bonds, and primarily affect interactions within the active site pocket formed by Asp 25/125 and Gly 271127. Some flexibility in the enzyme backbone at Gly 127 was also apparent. Based on these results, we suggest that the binding mode for central hydroxy-bearing, Cz-symmetric inhibitors will be determined by how well the inhibitor can simultaneously optimize hydrogen bonding with the active site carboxylate groups and van der Waals contacts with the neighboring backbone atoms of the active site "+"-loops. A symmetric hydrogenbonding arrangement with either one or two symmetrically positioned hydroxy groups appears to be preferred over less symmetric configurations.

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We report the first atomic resolution structure of an insect virus determined by single crystal X-ray diffraction. Black beetle virus has a bipartite RNA genome encapsulated in a single particle. The capsid contains 180 protomers arranged on a T = 3 surface lattice. The quaternary organization of the protomers is similar to that observed in the T = 3 plant virus structures. The protomers consist of a basic, crystallographically disordered amino terminus (64 residues), a beta-barrel as seen in other animal and plant virus subunits, an outer protrusion composed predominantly of beta-sheet and formed by three large insertions between strands of the barrel, and a carboxy terminal domain composed of two distorted helices lying inside the shell. The outer surfaces of quasi-threefold related protomers form trigonal pyramidyl protrusions. A cleavage site, located 44 residues from the carboxy terminus, lies within the central cavity of the protein shell. The structural motif observed in BBV (a shell composed of 180 eight-stranded antiparallel beta-barrels) is common to all nonsatellite spherical viruses whose structures have so far been solved. This highly conserved shell architecture suggests a common origin for the coat protein of spherical viruses, while the primitive genome structure of BBV suggests that this insect virus represents an early stage in the evolution of spherical viruses from cellular genes.

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HIV-1 protease is an effective target for designing drugs against AIDS, and structural information about the true transition state and the correct mechanism can provide important inputs. We present here the three-dimensional structure of a bi-product complex between HIV-1 protease and the two cleavage product peptides AETF and YVDGAA. The structure, refined against synchrotron data to 1.65 Å resolution, shows the occurrence of the cleavage reaction in the crystal, with the product peptides still held in the enzyme active site. The separation between the scissile carbon and nitrogen atoms is 2.67 Å, which is shorter than a normal van der Waal separation, but it is much longer than a peptide bond length. The substrate is thus in a stage just past the G'Z intermediate described in Northrop's mechanism [Northrop DB (2001) Acc Chem Res 34:790 -797]. Because the products are generated in situ, the structure, by extrapolation, can give insight into the mechanism of the cleavage reaction. Both oxygens of the generated carboxyl group form hydrogen bonds with atoms at the catalytic center: one to the OD2 atom of a catalytic aspartate and the other to the scissile nitrogen atom. The latter hydrogen bond may have mediated protonation of scissile nitrogen, triggering peptide bond cleavage. The inner oxygen atoms of the catalytic aspartates in the complex are 2.30 Å apart, indicating a low-barrier hydrogen bond between them at this stage of the reaction, an observation not included in Northrop's proposal. This structure forms a template for designing mechanism-based inhibitors.AIDS ͉ catalysis ͉ reaction intermediate ͉ x-ray crystallography H IV-1 protease (PR) is a homodimeric, aspartyl PR containing the signature sequence Asp-Thr-Gly in each monomer. The enzyme, which cleaves the viral polyprotein at eight different sites during the maturation process of the virus, is an important target for structure-based drug design (1-4). Emergence of drug-resistant mutations presents a new challenge, and additional inputs, such as the mechanism and interactions of the active enzyme with plain peptide substrates rather than with analogs, are required to tackle drug resistance. To address this question, we have undertaken to solve the crystal structures of HIV-1 PR oligopeptide substrate complexes. The enzyme used in the present study is a single polypeptide chain where the C terminus of the first monomer is linked with the N terminus of the second monomer through a pentapeptide (-GGSSG-) linker. The enzyme activity of such a tethered dimer (TD) construct is comparable with that of the native homodimer (5). Previously (6, 7), we have reported structures of TD and its complex with the undecapeptide substrate that corresponds in sequence to the reverse transcriptase-integrase junction in the viral polyprotein. Here we report the crystal structure of TD complexed with a decapeptide of amino acid sequence matching the reverse transcriptase-RNase H junction of the polyprotein. The structure has been refined to 1.65 Å resolution against diffra...

Structural snapshots of each step in the catalytic cycle would help development of inhibitors of human immunodeficiency virus type 1 protease (HIV-1 PR) as effective drugs against HIV/AIDS. We report here one snapshot obtained by determining the structure of enzyme-substrate complex under conditions where the catalytic activity of the enzyme is greatly reduced. The 1.76 A crystal structure shows the oligopeptide substrate, AETFYVDGAA, converted in situ into a gem-diol tetrahedral intermediate (TI). The gem-diol intermediate is neutral and one of the hydroxyl oxygens forms a very short hydrogen bond (2.2 A) with the anionic aspartate of the catalytic dyad, which is monoprotonated. Further, there is no hydrogen atom on the outer oxygen of the neutral aspartate. These two observations provide direct evidence that, in the reaction mechanism, hydrogen bonding between catalytic aspartate and scissile carbonyl oxygen facilitates water attack on the scissile carbon atom. Comparison with the structural snapshot of the biproduct complex involving the same substrate reveals the reorganization of the hydrogen bonds at the catalytic center as the enzymatic reaction progresses toward completion. Accumulation of TI in the crystals provides direct evidence that collapse of TI is the rate-limiting step of hydrolysis.

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