A rational approach towards successful crystallization and crystal treatment of human cytomegalovirus protease and its inhibitor complex

The crystallization and subsequent crystal treatment of both free human cyt omegalovirus (hCMV) protease and its inhibitor complexes are reported. For free-enzyme crystals, diffraction was greatly improved by optimizing the cr ystallization conditions, raising the precipitant concentration in the rese rvoir and soaking the crystals in artificial mother liquors. Each of the si x components in the final crystallization formula (16% PEG 4000, 0.1 M MES pH 6.0, 0.4 M LiCl, 10% glycerol, 5% t-butanol and 5 mM Na2S2O3) plays a di stinctive role and is indispensable. A synergistic effect of Na2SO4 and t-b utanol on diffraction was observed and studied. A 2.0 Angstrom multiwavelen gth anomalous diffraction (MAD) data set was collected using a synchrotron- radiation source, leading to the elucidation of the three-dimensional struc ture of the enzyme. For the inhibitor-complex crystals, initial attempts wi th co-crystallization and soaking experiments at pH 6.0 did not produce con clusive results. Subsequently, experiments were designed to co-crystallize the complex at pH 8.0, the optimal pH for the enzyme and the inhibitor acti vity. Using 20-50 mM spermine in the crystallization buffer, crystals of tw o peptidomimetic inhibitor complexes were obtained at pH 7.5 and 8.0. Sperm ine was required for the inhibitor complexes to be crystallized at pH 8.0, possibly neutralizing net negative charges of hCMV protease owing to its ac idic pI of 5.5. A 2.7 Angstrom data set was collected from one of the inhib itor complexes and the structure was determined using the molecular-replace ment method.