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 cytomegalovirus (hCMV) protease and its inhibitor complexes are reported. For free-enzyme crystals, diffraction was greatly improved by optimizing the crystallization conditions, raising the precipitant concentration in the reservoir and soaking the crystals in artificial mother liquors. Each of the six 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 distinctive role and is indispensable. A synergistic effect of Na₂SO₄ and t-­butanol on diffraction was observed and studied. A 2.0 Å multiwavelength anomalous diffraction (MAD) data set was collected using a synchrotron-radiation source, leading to the elucidation of the three-dimensional structure of the enzyme. For the inhibitor-complex crystals, initial attempts with co-crystallization and soaking experiments at pH 6.0 did not produce conclusive results. Subsequently, experiments were designed to co-crystallize the complex at pH 8.0, the optimal pH for the enzyme and the inhibitor activity. Using 20-50 mM spermine in the crystallization buffer, crystals of two peptidomimetic inhibitor complexes were obtained at pH 7.5 and 8.0. Spermine was required for the inhibitor complexes to be crystallized at pH 8.0, possibly neutralizing net negative charges of hCMV protease owing to its acidic pI of 5.5. A 2.7 Å data set was collected from one of the inhibitor complexes and the structure was determined using the molecular-replacement method.