Human cytomegalovirus (hCMV), a herpesvirus, infects up to 70% of the general population in the United States and can cause morbidity and mortality in immunosuppressed individuals (organ-transplant recipients and AIDS patients) and congenitally infected newborns. hCMV protease is essential for the production of mature infectious virions, as it performs proteolytic processing near the carboxy terminus (M-site) of the viral assembly protein precursor. hCMV protease is a serine protease, although it has little homology to other clans of serine proteases. Here we report the crystal structure of hCMV protease at 2.0 angstroms resolution, and show that it possesses a new polypeptide backbone fold. Ser 132 and His 63 are found in close proximity in the active site, confirming earlier biochemical and mutagenesis studies. The structure suggests that the third member of the triad is probably His 157. A dimer of the protease with an extensive interface is found in the crystal structure. This structure information will help in the design and optimization of inhibitors against herpesvirus proteases.

The binding site on the lymphocyte function-associated antigen-1 (LFA-1) of a class of hydantoin-based antagonists of leukocyte cell adhesion has been identified. This site resides in the inserted-domain (I-domain) of the CD11a chain at a location that is distal to residues known to be required for interactions with the intercellular adhesion molecules. This finding supports the hypothesis that the molecules are antagonizing cell adhesion via an allosteric modification of LFA-1. The binding site was identified using an integrated immunochemical, chemical, and molecular modeling approach. Antibodies that map to epitopes on the I-domain were blocked from binding to the purified protein by the hydantoins, indicating that the hydantoin-binding site resides on the I-domain. Photoaffinity labeling of the I-domain followed by LC/MS and LC/MS/MS analysis of the enzymatic digest identified proline 281 as the primary amino acid residue covalently attached to the photoprobe. Distance constraints derived from this study coupled with known SAR considerations allowed for the construction of a molecular model of the I-domain/inhibitor complex. The atomic details of the protein/antagonist interaction were accurately predicted by this model, as subsequently confirmed by the X-ray crystal structure of the complex.

Human cytomegalovirus (HCMV) protease belongs to a new class of serine proteases, with a unique polypeptide backbone fold. The crystal structure of the protease in complex with a peptidomimetic inhibitor (based on the natural substrates and covering the P4 to P1' positions) has been determined at 2.7 A resolution. The inhibitor is bound in an extended conformation, forming an anti-parallel beta-sheet with the protease. The P3 and P1 side chains are less accessible to solvent, whereas the P4 and P2 side chains are more exposed. The inhibitor binding mode shows significant similarity to those observed for peptidomimetic inhibitors or substrates of other classes of serine proteases (chymotrypsin and subtilisin). HCMV protease therefore represents example of convergent evolution. In addition, large conformational differences relative to the structure of the free enzyme are observed, which may be important for inhibitor binding.

A new polypeptide backbone fold for serine proteases has been identified based on the crystal structure ofhmnan cytomcgalovi.J.us protease. The structure was detem1ined at 2.5A resolution by the multiplewavelength anomalous diJ'fraction technique using the seleno-methionyl protein and refined at 2.0A resolution. It reveals a seven-stnmded mostly-antipm·allel 13-ban·eL which is sunounded by seven helices. The active site residues (Ser-132 and His-63) m·e situated on the outside of the 13-bmrel and in a groove on the surface of the protein. TI1e stmctme suggests that the third member of the catalytic triad is probably His-157. The protease of herpesviruses plays an essential role in the production of lllfectious virions 311d is an attractive tmget for the developement of antiherpes agents. The crystal stmcture information vvill help in the design m1d optimization of inhibitors agai.J.1st herpes virus protease.[1] Tong, L., Qian, C., Massariol, M. Human cytomegalovirus (HCMV) is a beta herpes vi.J.us. HCJ\.1Y, like all other members of the Herpes virus family, encodes a protease that is essential for capsid maturation and production of infectious virus. TI1e catalytic domain of the HCMV protease was produced in E.coli as a single-chain protein and was crystallized in space group C222 1 vvith two dimers per asymmetric unit. TI1e crystal stmcrure was deteffili.ned at 2.5A resolution using the ISAS method m1d noncryst:1llographico symmetry averaging. Om cmTent model has been refined against 2.3A data collected on an in1age plate at -170°C. The HCMV protease structure has a new fold different from that of any other 1.!1own protease. There is a central core comprising two orthogonal4-stranded beta sheets sunounded by eight alpha-helices. Residues in three flexible surface loops, including two associated with internal cleavage sites at ill11ino acids 143 and 209, have not been modeled into the wnent stmcture. Dimerization ofHCMV protease is mediated primarily by bmying four turns of alphaheli" (218-232) from one monomer into a pronounced depression in the smface of the other monomer. Only two of three residues previously in1plicated by ill11ino acid sequence alignment and mutagenesis asparticipating directly in catalysis (Ser-132 and His-63 but not Glu-122) me actually located in the active site. This novel structure is being used to finther understand the catalytic mechllilism. and to design inhibitors as potential anti-viral agents. Cysteine proteases related to mmnmalian inter!eukin-1~ convertlilg enzyme (ICE) and to its C. elegans homologue, CED-3, play a critical role in the biochemical events tl1at culminate in apoptosis. We have deteffili.ned the three-dimensional stmctme of a complex of tl1e hmnan CED-3 homologue CPP32/apopain witll a potent tetrapeptide-aldehyde inhibitor. I TI1e protein resembles ICE in overall structure, but its S 4 subsite is strikingly different in size and chemical composition. These differences account for the vmiation in specificity between tl1e ICE-and CED-3-related proteases and enable ...

Herpesvirus protease is required for the life cycle of the virus and is an attractive target for the design and development of new anti-herpes agents. The protease belongs to a new class of serine proteases, with a novel backbone fold and a unique Ser-His-His catalytic triad. Here we report the crystal structures of human cytomegalovirus protease in complex with two peptidomimetic inhibitors. The structures reveal a new hydrogen-bonding interaction between the main chain carbonyl of the P(5) residue and the main chain amide of amino acid 137 of the protease, which is important for the binding affinity of the inhibitor. Conformational flexibility was observed in the S(3) pocket of the enzyme, and this is supported by our characterization of several mutants in this pocket. One of the structures is at 2.5 A resolution, allowing us for the first time to locate ordered solvent molecules in the inhibitor complex. The presence of two solvent molecules in the active site may have implications for the design of new inhibitors against this enzyme. Favorable and stereospecific interactions have been established in the S(1)' pocket for one of these inhibitors.