Structure of granzyme C reveals an unusual mechanism of protease autoinhibition

Abstract

Proteases act in important homeostatic pathways and are tightly regulated. Here, we report an unusual structural mechanism of regulation observed by the 2.5-Å X-ray crystal structure of the serine protease, granzyme C. Although the active-site triad residues adopt canonical conformations, the oxyanion hole is improperly formed, and access to the primary specificity (S1) pocket is blocked through a reversible rearrangement involving Phe-191. Specifically, a register shift in the 190-strand preceding the activesite serine leads to Phe-191 filling the S1 pocket. Mutation of a unique Glu-Glu motif at positions 192-193 unlocks the enzyme, which displays chymase activity, and proteomic analysis confirms that activity of the wild-type protease can be released through interactions with an appropriate substrate. The 2.5-Å structure of the unlocked enzyme reveals unprecedented flexibility in the 190-strand preceding the active-site serine that results in Phe-191 vacating the S1 pocket. Overall, these observations describe a broadly applicable mechanism of protease regulation that cannot be predicted by template-based modeling or bioinformatic approaches alone.protease regulation ͉ register shift ͉ allostery P roteolysis plays an essential role in many biological pathways, an importance that is emphasized by the wide range and diversity of proteases that account for Ϸ2-3% of the human proteome (1). Of these, the largest group is the primary specificity (S1) family of serine proteases whose active sites contain three distinct features. The first is the catalytic triad (H57, D102, and S195 in chymotrypsin numbering) that creates a nucleophile to attack the substrate peptide bond. Mutation of any of these residues abolishes activity. Second is the ''oxyanion hole'' created by the main chain amide groups of S195 and G193. The positive charge in this region neutralizes a negatively charged oxygen atom in the substrate that is transiently formed during catalysis. Last, a conserved salt bridge is formed between the N-terminal amine group and the side chain of D194. This interaction introduces constraints on the active-site architecture that aid in cleavage efficiency.Given the diversity of irreversible effects that serine proteases can have on a variety of biological pathways, their actions are tightly regulated. During biosynthesis, most serine proteases follow a one-step activation pathway involving precursor processing. They are initially synthesized as inactive precursors (zymogens) in which the active site is unformed. Limited proteolysis at the N terminus removes the inactivating propeptide, thus allowing the protease to fold into a mature, active state. Once activated, serine proteases are controlled by a variety of inhibitors that may be either specific (for example, inhibitors of the serpin family) or nonspecific (such as ␣ 2 -macroglobulin) to ensure that they exert their effects only within the correct context. (3) or other proteins (4). This results in a two-step active-site assembly pathway for activation, from the ...

Conservation of the Extended Substrate Specificity Profiles Among Homologous Granzymes Across Species

“…Proteome-wide Substrate Specificity Profiles of GrH and Mutant GrC-The iceLogos (39) created for both proteases revealed an extended substrate specificity profile in line with previous data deduced from PS-SCL, phage display and Nterminal COFRADIC (16,19,20), with 86% of all human and mouse cleavage events after hydrophobic and/or aromatic amino acids (Fig. 3).…”

“…Recombinant, enterokinase activated wild-type GrC and the mutated form of GrC (E192R/E193G) were prepared as described previously (16). Recombinant human granzyme H was expressed in Pichia pastoris and purified to homogeneity.…”

“…94% of full length GrC and 99.6% of active GrC), furthermore showed that wild-type GrC can be restrained in its proteolytic function despite the presence of the catalytic triad residues His 57 -Asp 102 -Ser 195 (16). Comparing the crystal structures of GrC and GrA showed an unusual conformation of the active site, which could explain the inactivity of GrC.…”

“…Besides, in GrB-only knockout mice, a likely compensatory mechanism occurs, given that during cytotoxic lymphocyte activation, peak expression of GrC occurs earlier, giving rise to overall higher GrC levels as compared with wild-type mice (1,4,15). Of note, despite its implication in cytotoxicity, GrC was shown to be an inefficient cytotoxin, with a 2900-fold greater EC 50 value as compared with hGrB when delivered into P815 cells with recombinant mouse perforin (16).…”

Control of granzymes by serpins

“…Each additional paralogue has evolved a unique RCL implying non-redundant functions (Figure 4b), and as such the Serpinb9 RCL sequences should show similarity to the predicted substrate specificities of the additional granzymes. Indeed, many of the Serpinb9 RCLs contain bulky hydrophobic residues at the predicted P1, Chymase activity has been demonstrated for the mouse-specific granzyme C, 91 and is predicted for granzymes D, E, F, G and N. 92 In addition, the Pro-Leu motif in Serpinb9b suggests an interaction with gzmM. 93 Further investigations have shown that transfection with Serpinb9b does render cells resistant to exogenous recombinant gzmM.…”