Importance of the P4′ Residue in Human Granzyme B Inhibitors and Substrates Revealed by Scanning Mutagenesis of the Proteinase Inhibitor 9 Reactive Center Loop

Abstract

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 substrates. Here we have used the interaction of human granzyme B with a cognate serpin, proteinase inhibitor 9 (PI-9), to examine its substrate sequence requirements. Cleavage and sequencing experiments demonstrated that Glu340 is the P1 residue in the PI-9 RCL, consistent with the preference of granzyme B for acidic P1 residues. Ala-scanning mutagenesis demonstrated that the P4-P4′ region of the PI-9 RCL is important for interaction with granzyme B, and that the P4′ residue (Glu344) is required for efficient serpin-proteinase 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 (kcat/Km 9.5 × 103 and 1.2 × 105 s-1 M-1, respectively) but were not recognized by caspases. A substrate containing P1 Asp but lacking P4′ Glu was cleaved less efficiently (kcat/Km 5.3 × 10 4 s-1 M-1). An idealized substrate comprising the previously described optimal P4-P1 sequence (Ile-Glu-Pro-Asp) fused to the PI-9 P1′-P4′ sequence was efficiently cleaved by granzyme B (k cat/Km 7.5 × 105 s-1 M -1) and was also recognized by caspases. This contrasts with the literature value for a tetrapeptide comprising the same P4-P1 sequence (k cat/Km 6.7 × 104 s-1 M -1) and confirms that P′ residues promote efficient interaction of granzyme B with substrates. Finally, molecular modeling predicted that PI-9 Glu344 forms a salt bridge with Lys27 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 specific and efficient binding and propose that an acidic P4′ substrate residue allows discrimination between early (high affinity) and late (lower affinity) targets during the induction of apoptosis. Show more