Definition and function of structural determinants in granzyme B specificity

Trypsin-like serine proteases catalyze the hydrolysis of peptide bonds at specific extended amino acids sequences mediated by a constellation of active site amino acids that are still being characterized. Complete understanding of the relationship between contacting amino acids, substrate specificity and efficient catalysis will enable prediction of substrate specificity from genomic sequence data.;To probe structural determinants of substrate specificity the serine protease granzyme B was studied. Granzyme B is unique among mammalian proteases, requiring an aspartic acid adjacent to the scissile bond, and a specific extended substrate encompassing six amino acids for efficient hydrolysis. The x-ray crystallographic solution of rat granzyme B [N66Q] in complex with ecotin [81--84 IEPD] exposed ten structural determinants of specificity. The active site, when compared with trypsin, included a truncated binding site loop, a deleted disulfide bond and a buried primary specificity determinant, Arg226, mediating aspartic acid specificity. Site directed mutagenesis and kinetics analysis of trypsin variants concluded that inserting Arg226 and mutations to both of the active site loops were required for acidic specificity. Trypsin DS(-) ARR preferentially hydrolyzed substrates containing aspartic acid with 10-6 wild type activity. Determinants of granzyme B extended specificity identified from the crystal structure were investigated with site-directed mutagenesis and synthetic combinatorial substrate libraries. Binding site variants firmly established the protease residues in direct contact with the substrate as the primary extended specificity determinants, and variations effected one subsite profile at a time. Mutation of structural determinants alters subsite specificity and catalytic efficiency by changing hydrogen bond, steric and hydrophobic effects. Importantly, the specificity of a single subsite was exchanged between granzymes B and C by the mutation of one structural determinant. The structural determinants of granzyme B mediated the substrate specificity, but did not identify all of the amino acids responsible for its remarkable specificity. Future design of substrate specificity should focus on individually modifying the extended specificity, and discovering additional structural determinants.