Human herpesviruses are responsible for a variety of diseases. They are divided into three subfamilies: alpha includes herpes simplex viruses (HSV-1 and HSV-2) and varicella-zoster virus (VZV); beta includes cytomegalovirus (CMV) and human herpesvirus-6 (HHV-6); and gamma includes Epstein-Barr virus (EBV). Each virus encodes a serine protease that is essential for its replication and is a potential target for therapeutic intervention. Human CMV is a ubiquitous opportunistic pathogen that can result in life-threatening infections in congenitally infected infants, immunocompromised individuals and immunosuppressed cancer or transplant patients. Here we report the crystal structure of human CMV protease at 2.5 angstroms resolution. The structure reveals a fold that has not been reported for any other serine protease, and an active site consisting of a novel catalytic triad in which the third member is a histidine instead of an aspartic acid, or possibly a catalytic tetrad consisting of a serine, two histidines and an aspartic acid. An unusual dimer interface that is important to the protease activity has also been identified.

The 3.0-resolution x-ray structure of rat liver 3a-hydroxysteroid dehydrogenase/dihydrodiol dehydrogenase (3a-HSD, EC 1.1.1.50) was determined by molecular replacement using human placental aldose reductase as the search model. The protein folds into an a/ or triose-phosphate Isomerase barrel and lacks a coI Rossmann fold for bnding pyridine nucleotide. The structure contains a concentration of hydrophobic amino adds that lie in a cavity near the top ofthe barrel and that are presumed to be involved in binding hydrophobic substrates (steroids, prostaglandins, and polycyclic aromatic hydrocarbons) and inhibitors (nonsteroidal antdinflammatory drugs). At the distal end of this cavity lie three residues in close proximity that have been implicated in catalysis by site-directed mutagens-Tyr-55, Tyr-55 is postulated to act as the general acid. 3a-HSD shares significant sequence identity with other HSDs that belong to the aldo-keto reductase superfamily and these may show similar architecture. Other members of this family include prostaglandin F synthase and p-crystallin. By contast, 3a-HSD shares no sequence identity with HSDs that are members ofthe short-chain alcohol dehydrogenase family but does contain the Tyr-XaaXaa-Xaa-Lys consensus sequence imlated in catalysis in this family. In the 3a-HSD structure these residues are on the periphery of the barrel and are unlikely to participate in catalysis.3a-Hydroxysteroid dehydrogenase [3a-HSD; 3a-hydroxysteroid:NAD(P)+ oxidoreductase, EC 1.1.1.50] from rat liver cytosol is a monomeric NAD(P)+-linked oxidoreductase with a molecular mass of 37,029 daltons deduced from its cDNA clone (1). The enzyme reduces biologically important 3-oxo steroids of the androstane (C19), pregnane (C21), and cholane (C24) series, in which the A/B ring fusion may be in the cis or trans configuration (2). These reactions lead to the inactivation of circulating androgens, progestins, and glucocorticoids as well as the biosynthesis of bile acids (3). The enzyme functions as a dihydrodiol dehydrogenase and, by oxidizing trans-dihydrodiols of polycyclic aromatic hydrocarbons, can suppress the formation of the ultimate carcinogens: the anti-diol epoxides (4). The enzyme also displays 9-, 11-, and 15-hydroxyprostaglandin dehydrogenase activity (5). This multifunctional enzyme is potently inhibited by nonsteroidal antiinflammatory drugs and can be used to screen the antuinflammatory potency of unknowns (6). Structural information is required to understand how this enzyme recognizes its diverse ligands. 3a-HSD shares high amino acid sequence identity with bovine lung prostaglandin F synthase (69%), human chlordecone reductase (66%), and frog lens p-crystallin (55%), which are members of the aldo-keto reductase superfamily. Included in this family are human placental, bovine, and rat lens aldose reductase, which share 58% sequence identity with 3a-HSD (ref. 1 and references therein). Two newly sequenced HSDs which belong to this family include 20a-HSD from rabbit ovary (7) and bovine testis (8)...

Human herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are responsible for herpes labialis (cold sores) and genital herpes, respectively. They encode a serine protease that is required for viral replication, and represent a viable target for therapeutic intervention. Here, we report the crystal structures of HSV-1 and HSV-2 proteases, the latter in the presence and absence of the covalently bound transition state analog inhibitor diisopropyl phosphate (DIP). The HSV-1 and HSV-2 protease structures show a fold that is neither like chymotrypsin nor like subtilisin, and has been seen only in the recently determined cytomegalovirus (CMV) and varicella-zoster virus (VZV) protease structures. HSV-1 and HSV-2 proteases share high sequence homology and have almost identical three-dimensional structures. However, structural differences are observed with the less homologous CMV protease, offering a structural basis for herpes virus protease ligand specificity. The bound inhibitor identifies the oxyanion hole of these enzymes and defines the active site cavity.

To gain greater understanding of the structural basis of human immunodeficiency virus (HIV) protease ligand specificity, we have crystallized and determined the structures of the HIV-1 protease (Val32Ile, Ile47Val, Val82Ile) triple mutant and simian immunodeficiency virus (SIV) protease in complex with SB203386, a tripeptide analogue inhibitor containing a C-terminal imidazole substituent as an amide bond isostere. SB203386 is a potent inhibitor of HIV-1 protease (Ki = 18 nM) but shows decreased inhibition of the HIV-1 protease (Val32Ile, Ile47Val, Val82Ile) triple mutant (Ki = 112 nM) and SIV protease (Ki = 960 nM). Although SB203386 binds in the active site cavity of the triple mutant in a similar fashion to its binding to the wild-type HIV-1 protease [Abdel-Meguid et al. (1994) Biochemistry 33, 11671], it binds to SIV protease in an unexpected mode showing two inhibitor molecules each binding to half of the active site. Comparison of these two structures and that of the wild-type HIV-1 protease bound to SB203386 reveals that HIV protease ligand specificity is imparted by residues outside of the catalytic pocket, which causes subtle changes in its shape. Furthermore, this work illustrates the importance of structural studies in order to understand the structure-activity relationship (SAR) between related enzymes.

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