Importance of the P4 ' residue in human granzyme B inhibitors and substrates revealed by scanning mutagenesis of the proteinase inhibitor 9 reactive center loop

The cytotoxic lymphocyte serine proteinase granzyme B induces apoptosis of abnormal cells by cleaving intracellular proteins at sites similar to those cleaved by caspases, Understanding the substrate specificity of granzyme B will help to identify natural targets and develop better inhibitors or sub strates, Here we have used the interaction of human granzyme B with a cogna te serpin, proteinase inhibitor 9 (PI-9), to examine its substrate sequence requirements. Cleavage and sequencing experiments demonstrated that Glu(34 0) is the P1 residue in the PI-9 RCL, consistent with the preference of gra nzyme B for acidic P1 residues. Ale-scanning mutagenesis demonstrated that the P4-P4' region of the PI-9 RCL is important for interaction with granzym e B, and that the P4' residue (Glu(344)) is required for efficient serpin-p roteinase binding. Peptide substrates based on the P4-P4' PI-9 RCL sequence and containing either P1 Glu or P1 Asp were cleaved by granzyme B (k(cat)/ K-m 9.5 x 10(3) and 1.2 x 10(5) s(-1) M-1, respectively) but were not recog nized by caspases, A substrate containing P1 Asp but lacking P4' Glu was cl eaved less efficiently (k(cat)/K-m 5.3 x 10(4) s(-1) M-1). An idealized sub strate comprising the previously described optimal P4-P1 sequence (Ile-Glu- Pro-Asp) fused to the PI-9 P1'-P4' sequence was efficiently cleaved by gran zyme B (k(cat)/K-m 7.5 x 10(5) s(-1) M-1) and was also recognized by caspas es. This contrasts with the literature value for a tetrapeptide comprising the same P4-P1 sequence (k(cat)/K-m 6.7 x 10(4) s(-1) M-1) and confirms tha t P' residues promote efficient interaction of granzyme B with substrates. Finally, molecular modeling predicted that PI-9 Glu(344) forms a salt bridg e with Lys(27) Of granzyme B, and we showed that a K27A mutant of granzyme B binds less efficiently to PI-9 and to substrates containing a P4' Glu. We conclude that granzyme B requires an extended substrate sequence for speci fic and efficient binding and propose that an acidic P4' substrate residue allows discrimination between early thigh affinity) and late (lower affinit y) targets during the induction of apoptosis.