A new serine-protease fold revealed by the crystal structure of human cytomegalovirus protease

Abstract

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.

Chemical Rescue of I-site Cleavage in Living Cells and in Vitro Discriminates between the Cytomegalovirus Protease, Assemblin, and Its Precursor, pUL80a

“…It has also been suggested that without R-site cleavage, conformation of the pPR sequence destined to form the carboxyl terminus of assemblin would be different (14) and possibly unable to participate in the interactions and changes, which accompany dimerization and activation. Thus, one explanation of our finding is that imidazole rescue of I-site cleavage works only when the active site residues are in the relative orientation they assume in the activated assemblin dimer, which may be different in the precursor.…”

“…The crystal structure of assemblin places the I site within an unstructured loop in the vicinity of the active site (11)(12)(13)(14). Extension of the loop toward the interior of the protease would situate the I site in proximity to the catalytic residues.…”

Experiences from the Structure Determination of Human Cytomegalovirus Protease

“…This could lead to a tighter packing of the protease molecules. Second, sodium sulfate caused a small change in the organization of the HCMV protease dimer in these crystals (Tong et al, 1996). This might also have an impact on the crystal packing, as evidenced by the fact that crystal form D has the shortest c-axis length.…”

“…The amino-acid sequences of herpesvirus proteases have no detectable homology to other serine proteases or hydrolases. The crystal structure of HCMV protease has recently been determined in our laboratory by the seleno-methionyl multiple-wavelength anomalous diffraction (MAD) technique (Tong et al, 1996), and in other laboratories by the heavy-atom isomorphous replacement technique (Qiu et al, 1996;Shieh et al, 1996;Chen et al, 1996). It showed that HCMV protease possesses a new polypeptide backbone fold; confirmed that Ser132 and His63 are in close proximity in space; and identified His157 as the possible third member of the catalytic triad.…”

“…The current atomic model, after structure refinement at 2.0A resolution (Table 4), is still missing about 40 residues of each monomer of the protease. These residues are in regions of very low sequence homology among the herpesvirus proteases and may be disordered in structure (Tong et al, 1996).…”