MMP-2 is a member of the matrix metalloproteinase family that has been implicated in tumor cell metastasis and angiogenesis. Here, we describe the solution structure of a catalytic domain of MMP-2 complexed with a hydroxamic acid inhibitor (SC-74020), determined by three-dimensional heteronuclear NMR spectroscopy. The catalytic domain, designated MMP-2C, has a short peptide linker replacing the internal fibronectin-domain insertion and is enzymatically active. Distance geometry-simulated annealing calculations yielded 14 converged structures with atomic root-mean-square deviations (r.m.s.d.) of 1.02 and 1.62 Å from the mean coordinate positions for the backbone and for all heavy atoms, respectively, when 11 residues at the N-terminus are excluded. The structure has the same global fold as observed for other MMP catalytic domains and is similar to previously solved crystal structures of MMP-2. Differences observed between the solution and the crystal structures, near the bottom of the S1′ specificity loop, appear to be induced by the large inhibitor present in the solution structure. The MMP-2C solution structure is compared with MMP-8 crystal structure bound to the same inhibitor to highlight the differences especially in the S1′ specificity loop. The finding provides a structural explanation for the selectivity between MMP-2 and MMP-8 that is achieved by large inhibitors.

HERPESVIRUSES encode a serine protease(1,2) that specifically cleaves assembly protein(3). This protease is critical for replication(4), and represents a new target for antiviral drug design(5). Here we report the three-dimensional structure of the protease from human cytomegalovirus (hCMV) at 2.27 Angstrom resolution. The structure reveals a unique fold and new catalytic strategy for cleavage. The monomer fold of the enzyme, a seven-stranded beta-barrel encircled by a chain of helices that form the carboxy terminus of the molecule, is unrelated to those observed in classic serine proteases such as chymotrypsin and subtilisin. The serine nucleophile at position 132 is activated by two juxtaposed histidine residues at positions 63 and 157. Dimerization, which seems to be necessary for activity(6,7), is observed in the crystals. Correlations of the structure with the sequences of herpesvirus proteases(1,5,8) suggest that dimerization may confer specificity and recognition in substrate binding.

The crystallization of homogeneous or highly purified macromolecules depends on many variables such as precipitant, pH, choice of buffer, protein concentration, temperature, the participation of different mono- and divalent ions, as well as the presence of minute amounts of detergent and organic molecules. Finding the best combination among these many parameters is a multi-variable optimization problem. This kind of problem can be treated mathematically by sampling techniques. We have used this technique for protein crystallization. The iterative procedure starts with random sampling, followed by quantitative evaluation and cycles with weighted sampling. A simple procedure, derived from this concept and called MON48, has been successfully applied to many protein crystallization problems.