Structural Basis for Proteolytic Specificity of the Human Apoptosis-Inducing Granzyme M

Lianfeng Wu, Li Wang, Guoqiang Hua, Kan Liu, Xuan Yang, Yujia Zhai, Mark Bartlam, Fei Sun and Zusen Fan
J Immunol July 1, 2009, 183 (1) 421-429; DOI: https://doi.org/10.4049/jimmunol.0803088

Abstract

Granzyme M (GzmM), a unique serine protease constitutively expressed in NK cells, is important for granule-mediated cytolysis and can induce rapid caspase-dependent apoptosis of tumor cells. However, few substrates of GzmM have been reported to date, and the mechanism by which this enzyme recognizes and hydrolyzes substrates is unknown. To provide structural insights into the proteolytic specificity of human GzmM (hGzmM), crystal structures of wild-type hGzmM, the inactive D86N-GzmM mutant with bound peptide substrate, and the complexes with a catalytic product and with a tetrapeptide chloromethylketone inhibitor were solved to 1.96 Å, 2.30 Å, 2.17 Å and 2.70 Å, respectively. Structure-based mutagenesis revealed that the N terminus and catalytic triad of hGzmM are most essential for proteolytic function. In particular, D86N-GzmM was found to be an ideal inactive enzyme for functional studies. Structural comparisons indicated a large conformational change of the L3 loop upon substrate binding, and suggest this loop mediates the substrate specificity of hGzmM. Based on the complex structure of GzmM with its catalytic product, a tetrapeptide chloromethylketone inhibitor was designed and found to specifically block the catalytic activity of hGzmM.

Granzyme (Gzm)4 -induced cell death is a major pathway used by CTL and NK cells to eliminate virus-infected or transformed tumor cells (1, 2). Gzms are normally expressed in an inactive prostate called Pro-Gzm, and the N terminus of Pro-Gzm is subsequently cleaved to release the active form with the constitutive N-terminal sequence IIGG. Five types of human Gzms (A, B, H, K, and M) have been identified to date. GzmA and B are the most abundant Gzms in CTLs and lymphokine-activated killer cells and have been extensively studied (3, 4, 5, 6, 7).

GzmM, a chymotrypsin-like serine protease, preferentially cleaves its substrate after Met or Leu (8, 9). Human Gzm (hGzm)M is encoded in a distinctive cluster on chromosome 19 and colocalizes with a family of neutrophil elastases (10). GzmM is constitutively and highly expressed in activated NK cells, but is never detected in CD4+ or CD8+ T cells even after activation (11, 12). GzmM plays very important roles in granule-mediated cytolysis and can induce rapid cell death via an as yet undefined mechanism (13, 14). Several substrates have been identified for GzmM so far, including the GzmB serpin PI9 (15), the inhibitor of caspase-activated DNase (14), the reactive oxygen species antagonist TRAP1 (16), the component of cytoskeleton α-tubulin (17), and the abundant nucleolar phosphoprotein nucleophosmin (18).

Human Gzms share high sequence homology and similar structures. However, positional screening techniques determined that they possess distinctive substrate specificities (8). High-resolution structures of the Gzms are indispensable for further investigation of their specific substrate binding sites. To date, the three-dimensional structures of human GzmA, B, and Pro-GzmK are known and have revealed the structural basis for their substrate recognition (19, 20, 21, 22), yet no structures are available for hGzmM and hGzmH.

To elucidate the substrate-binding specificity and catalytic mechanisms of GzmM, we have determined the crystal structures of wild-type hGzmM, the inactive D86N-GzmM mutant bound with a peptide substrate (D86N-Sub), hGzmM in complex with a catalytic product (aM-Prod), and hGzmM in complex with a tetrapeptide CMK inhibitor (aM-Inhibitor) to 1.96 Å, 2.30 Å, 2.17 Å, and 2.70Å, respectively. Based on our structural analysis, we generated a series of mutants to characterize the determinants of hGzmM enzymatic activity and found that Asp86 and His41 in the catalytic triad contribute more to proteolytic activity than the attack residue Ser182. We also found the D86N-GzmM mutant is an ideal catalytically inactive (dead) enzyme for functional studies. From structural comparisons, we observed a large conformational change of the L3 loop upon substrate binding and found this loop could endow the substrate with recognition and specificity. Based on the complex structure with a catalytic product, we designed a tetrapeptide CMK inhibitor and found it can specifically block the catalytic activity of hGzmM.

Materials and Methods

Plasmid construction for hGzmM and its variants

The cDNA fragments of Pro-hGzmM, active hGzmM, and the truncated hGzmM (ΔII-GzmM) were amplified from the full length cDNA of hGzmM (RZPD German Resource Center for Genome Research) by standard PCR cloning strategies. The other mutations were generated by site-directed mutagenesis using the Phusion DNA polymerase kit (New England Biolabs). The enterokinase cleavage sequence (DDDDK) was introduced directly before the N terminus of each target protein and the exact N terminus (IIGG) was released after enterokinase cleavage (the construction strategies are shown in Fig. 2B). All segments were subcloned into the pET-26b vector (Novagen).

Expression and purification of recombinant proteins

All GzmM variant proteins were expressed in Escherichia coli and refolded from inclusion bodies according to a previous protocol (21). The Rosetta (DE3) cell strain was used to express GzmM and its variants. The harvested pellets were lysed in a lysis buffer (50 mM Tris, 500 mM NaCl, 0.25 mg/ml lysozyme, 10 μg/ml RNase A, 5 μg/ml DNase I, 2 mM MgCl2, 0.1% Triton X-100, pH 7.9). The inclusion bodies were prepared and dissolved overnight in a buffer (6 M guanidinium chloride, 100 mM Tris-HCl, 20 mM EDTA, 150 mM GSH, and 15 mM GSSG, pH 8.0). They were refolded in a refolding buffer (0.5 M Tris-HCl, 0.5 M l-arginine, 20 mM CaCl2, 0.1 M NaCl, and 0.5 mM l-cysteine, pH 8.5) at 4°C according to the reported protocol. After adequate refolding, the proteins were dialyzed into the binding buffer (50 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole 10% glycerol, pH 8.0) and purified by Ni-NTA columns. Subsequently, the enriched proteins were treated by enterokinase (Novagen) in 50 mM Tris-HCl, 2 mM CaCl2, pH 8.0 at 18°C. Finally, the treated proteins were purified with a Resource S column (GE Healthcare) to exclude enterokinase. rTRAP1, rSET, and GST-Bid were expressed in E. coli and purified as described previously (16, 23).

Crystallization and data collection

The purified proteins were concentrated to 15 mg/ml in a buffer containing 20 mM Tris-HCl, pH 7.9 for crystallization screening experiments. Crystals of hGzmM were grown in 0.2 M Li2SO4, 0.1 M Tris, and 25% w/v PEG 3350, pH 8.5. The D86N-GzmM mutant enzyme was mixed with an octapeptide substrate (SSGKVPLS) in a molar ratio (1:5) in a buffer containing 20 mM Tris-HCl, pH 8.0 and crystallized in the same condition as active hGzmM. The octapeptide substrate or inhibitor was soaked into the active GzmM crystals to achieve the two complex crystals (aM-Prod and aM-Inhibitor). All crystallization experiments were performed using the hanging drop vapor diffusion method at 16°C. Before data collection, crystals were soaked in cryo-protectant solution containing 0.1 M Bicine, 0.2 M Li2SO4, 30% PEG 3350, pH 8.0 for 30 s, followed by direct flash-cooling in a liquid nitrogen cryostream. Diffraction data for the active hGzmM and aM-Inhibitor were collected on beam line 3W1A of the Beijing Synchrotron Radiation Facility using x-rays of wavelength 0.9794 Å at 95 K. Data for the D86N-Sub and aM-Prod complexes were collected in house (Rigaku FR-E x-ray generator, R-Axis IV++ image plate detector, λ = 1.5418 Å) at 93 K. With the exception of diffraction data for the aM-Inhibitor complex, which were processed by Mosflm 7.0.3 (24), all other diffraction data were processed by HKL2000 (25). Data processing statistics are summarized in Table I⇓.

View this table:
Table I.

Data collection, phasing, and refinement statisticsa

Structure determination and refinement

The molecular replacement method was used to determine the structure of active hGzmM. An initial search model was generated from the coordinates of human complement factor D (Protein Data Bank entry 1DST) with the program Modeler7 (26). Phaser (27) was used to find the unique top solution in the rotation and translation function and to calculate the optimal phases. ARP/wARP (28) was then used for automatic model building with 99% completeness. COOT (29) was used to refine the model manually, combined with interactive restrained refinement by Refmac5.0 (30). The final model was refined to 1.96 Å resolution with Rwork = 20.9% and Rfree = 25.7%. All complex structures were solved by the molecular replacement method using the active hGzmM structure and refined with the translation/libration/screw motion (TLS) and restrained refinement method using Refmac5.0. All final models were judged to have good stereochemistry from Ramachandran plots calculated by PROCHECK (31). Structure refinement statistics are shown in Table I⇑. All figures for surfaces, ribbons, balls, and sticks were generated with PYMOL (http://pymol.sourseforge.net) and BobScript 2.6b (32).

Proteolysis assays

The enzymatic activity of hGzmM was detected by cleavage of a synthetic substrate, Suc-AAPL-pNA, labeled with fluorescence groups (pNA) at the C terminus, 0.5 μM active GzmM or its variants were incubated with the substrate (300 μM) at 37°C for 2 h and the fluorescence signal was monitored at 405 nm with a Multilabel Counter (Wallac 1420 Victor; PerkinElmer). For the rTRAP1 cleavage assay, 1.5 μM rTRAP1 was incubated with 1.5-μM active GzmM or its variants at 37°C for 2 h. The products were analyzed by SDS-PAGE. All data were measured from at least three separate experiments.

Loading assay

To further investigate the physiological relevance of hGzmM and its variants, they were loaded into Jurkat cells with an optimal dose of adenovirus at 37°C for 4 h as previously described (14). Briefly, Jurkat cells (2 × 105) were washed three times in HBSS and resuspended in loading buffer (HBSS with 0.5 mg of BSA per ml, 1 mM CaCl2, 1 mM MgCl2). The resuspended cells were then treated with 1.5 μM hGzmM or its variants plus an optimal concentration of adenovirus at 37°C for 4 h. Treated cells were double-stained with Annexin V-Fluos (recombinant human Annexin V conjugated with FITC; Bender MedSystems) and propidium iodide (PI) and followed by flow cytometry (FACSCalibur; BD Biosciences). The data were analyzed by CellQuest software.

Results

Overall structure of human GzmM

Recombinant hGzmM was expressed as inclusion bodies in E. coli, but could be successfully refolded with strong enzymatic activity. Purified hGzmM was crystallized in the P3121 space group with one molecule per asymmetric unit and its crystal structure was solved to 1.96 Å by molecular replacement. The overall structure, containing four α-helices and 13 β-strands, is separated into two domains. The first domain (domain N) is comprised of seven β-strands (β1 to β7) and three α-helices (α1, α2, and α4). The second domain (domain C) contains six β-strands (β8 to β13) and one α-helix (α3) (Fig. 1⇓). Domains N and C are largely connected by two loops, L1 (residues 1–15) and L2 (residues 99–124), and the catalytic triad (Asp86, His41, and Ser182) is located in the cleft between domains akin to GzmA and B (19, 20, 22). Loop L2 joins the two domains and is usually referred to as the “autolysis loop” (21). Loop L1 effectively hooks domain N by inserting its conserved N-terminal tail into the hydrophobic pocket of domain C. The nonconserved loop L3 (residues 199–212), close to the catalytic triad, protrudes into the molecular surface and makes an important contribution to the specificity of substrate recognition, as described below. Four disulfide bonds, DS1 (C26–C42), DS2 (C120–C188), DS3 (C151–C167), and DS4 (C178–C205), help to maintain the overall stability of the structure.

FIGURE 1.
FIGURE 1.

Stereo view of the active hGzmM structure. β-strands (β1 to β13) are labeled and presented in cyan for domain N and slate blue for domain C. α-helices (α1 to α4) are labeled and colored in magenta. Three loops are depicted as L1 (1–15, dark blue), L2 (99–124, orange) and L3 (199–212, red). Four pairs of disulfide bonds (DS1, DS2, DS3, and DS4) are colored with carbon atoms in yellow and sulfur atoms in green. The catalytic triad is displayed as ball-and-stick and labeled. The N and C terminus are indicated with characters (N and C). In this text, if not declared, all oxygen atoms are colored by red and nitrogen atoms by blue.

The N-terminal hook is important for GzmM stability and activity

Active Gzm proteins, with a few exceptions, possess a highly conserved hydrophobic N-terminal IIGG sequence that turns in toward the interior of the structure after activation (19, 33, 34). In the case of hGzmM, the N-terminal tail twists at Gly3 and forms strong interactions with the surrounding pocket (Fig. 2⇓A), including hydrophobic interactions among Ile1, Ile2, and the pocket; a hydrogen bond between the nitrogen of Ile1 and carboxyl oxygen of Asp181; a hydrogen bond between the carbonyl oxygen of Ile2 and the amide nitrogen of Ala176; and several bridges mediated by water molecules. The importance of the N-terminal conformation has previously been addressed in chymotrypsin (35), but more systematic data for hGzmM are provided here. Based on structural analysis, several variants were constructed with an enterokinase cleavage sequence to exclude redundant residues from the N terminus (Fig. 2⇓B). The Gly3 mutated to Pro (G3P-GzmM) mutant and ΔII-GzmM (Ile1 and Ile2 deleted), in which the N terminus insertion structure has been abolished, were highly unstable and quickly degraded. Compared with active wild-type hGzmM, the mutants I1,2L-GzmM (Ile1 and Ile2 mutated to Leu), I1,2A-GzmM (Ile1 and Ile2 mutated to Ala), and the proenzyme Pro-hGzmM (with an additional propeptide SSFGTQ before the active N terminus of hGzmM) showed greatly decreased activity when cleaving the peptide substrate or the rTRAP1 protein substrate (Fig. 2⇓, C and D). In particular, I1,2A-GzmM underwent a complete loss of catalytic activity. It is evident that mutation of Ile to Leu will negatively influence the hydrophobic interactions between the N terminus and its surrounding pocket, while mutation of Ile to Ala will greatly decrease this interaction. These data indicate that any modification to the N terminus of active hGzmM could abolish its stability or catalytic activity.

FIGURE 2.
FIGURE 2.

The N-terminal insertion is critical for catalytic activity of hGzmM. A, Ribbon plot and surface representations illustrating the structure of the N-terminal insertion. Hydrophobic, hydrophilic, negative, and positive surfaces are indicated by white, green, red, and blue, respectively. Four N-terminal residues are labeled and displayed as ball-and-stick. B, Scheme for the expression strategies of GzmM variants. All GzmM variants were initially expressed with enterokinase (EK) recognition sites, which were removed from their N terminus after purification with Ni-NTA column. Expression strategies for Pro-GzmM, active GzmM (aM), and I1,2L- and I1,2A-GzmM are demonstrated in the scheme. Moreover, other mutations involved in this study are indicated with ∗ in the plot. C, Substrate hydrolysis activity for hGzmM and its variants detected by the fluorescence peptide substrate. ProM, Pro-GzmM; I1,2L, I1,2L-GzmM; I1,2A, I1,2A-GzmM; D181N, D181N-GzmM. D, Proteolytic activity assay for hGzmM and its variants by cleavage of rTRAP1. M, molecular marker. E, Location relationship between the N terminus and catalytic pocket of hGzmM. All related residues are labeled and shown in ball-and-stick representation, and their surrounding environment is displayed in ribbon representation. N-terminal residues are colored with green carbon atoms and catalytic residues with cyan carbon atoms. Hydrogen bonds are shown with gray dashed lines and the Ile1-Asp181 hydrogen bond with a blue dashed line. Those regions interacting with the N terminus are colored purple and the substrate binding sites are colored green.

The catalytic triad and the catalytically inactive mutant D86N-GzmM

The catalytic triad of hGzmM is composed by Asp86, His41, and Ser182 (Fig. 2⇑E), similar to many classical serine proteases. Ser182, whose acidity is strengthened by Asp86 via His41 through hydrogen bonds, will attack and cleave the substrate directly. As in previous reports, all three residues are clearly important for the proteolytic activity of hGzmM, which we further confirmed by site-directed mutagenesis. The H41N-GzmM, D86N-GzmM, and S182A-GzmM mutants were expressed in E. coli and refolded from inclusion bodies as previously described. From in vitro proteolytic activity assays involving cleavage of the hGzmM peptide substrate or the rTRAP1 protein substrate, S182A-GzmM appeared to retain only 5–10% of the wild-type enzymatic activity, while H41N-GzmM showed little activity and D86N-GzmM completely lost its proteolytic activity (Fig. 3⇓, A and B). Similar results were observed in the kinetic parameter measurement assays; the activities of H41N- and D86N-GzmM could not be detected within the 40 min time limit, whereas S182A-GzmM exhibited little activity (data not shown).

FIGURE 3.
FIGURE 3.

Proteolytic determinants of the catalytic triad. A, Substrate hydrolysis activity for hGzmM and its variants detected by the fluorescence peptide substrate. H41N, H41N-GzmM; D86N, D86N-GzmM; S182A, S182A-GzmM. B, Proteolytic activity for hGzmM and its variants by cleavage of rTRAP1. M, molecular marker. C, pH dependent substrate hydrolysis activity for hGzmM. D, Proteolytic activity for hGzmA and its variants by cleavage of rSET. aA, active GzmA; S195A, S195A-GzmA; D102N, D102N-GzmA.

Proteolytic activity assays suggest that Asp86 makes a greater contribution to the enzymatic activity than does Ser182, indicating that the acidity of Asp86 provides the major driving power for catalysis. We further confirmed this by a pH-dependent enzyme activity assay (Fig. 3⇑C), from which the enzymatic activity of hGzmM gradually increased as the pH was increased from 5.0 to 9.0 and reached its maximum at pH 8.0. These results indicate that Asp86 can readily lose its carboxyl proton under basic conditions to promote formation of His41•H+ and greatly strengthen the cleavage of the Ser182-O− scissile bond. Taken together, Asp86 is the most dominant residue in the catalytic triad. Mutation of Asp86 to Asn86 will result in a loss of the catalytic driving power and completely abolish the enzymatic activity of hGzmM.

When performing functional studies of Gzm enzymes, researchers usually create a catalytically inactive enzyme by mutating the attacking Ser residue to Ala. However, it is difficult to completely abolish the proteolytic activities for these mutants. For example, the S182A-GzmM mutant retained 5–10% enzymatic activity and could still exert its roles in cleaving substrates (Fig. 3⇑, A and B) and inducing cell death (unpublished data). Similar results were also observed for GzmA (Fig. 3⇑D) and reported for GzmH (36).

Despite being catalytically inactive, the structure of the D86N-GzmM mutant (data not shown) is identical with that of the wild-type enzyme. This indicates that the D86N-GzmM mutant can be used as a catalytically dead enzyme for functional studies. To verify whether the Asp mutation results in a catalytically inactive enzyme in other Gzms, D102N-GzmA and S195A-GzmA mutants were generated and their proteolytic activities were assayed as described (3). Compared with active GzmA in degrading rSET experiments, S195A-GzmA appeared to possess clear proteolytic activity, whereas D102N-GzmA showed no enzymatic activity at all (Fig. 3⇑D). These results indicate that GzmM and GzmA mutants, in which Asp is changed to Asn, are catalytically dead enzymes. This may also hold true for other Gzm enzymes.

Determinants of substrate recognition and substrate specificity

To investigate the detailed interaction between hGzmM and its substrate, the octa-peptide (OPEP) SSGKVPLS was synthesized according to previous work (15), and the complex structure (D86N-Sub) between D86N-GzmM and OPEP was solved to 2.30 Å. At the same time, OPEP was used to soak crystals of wild-type hGzmM. The resulting complex structure (aM-Prod) between wild-type hGzmM and the cleaved peptide (CPEP) SSGKVPL was solved to 2.17 Å. The bound OPEP and CPEP were clearly identified and traced in 2Fo-Fc omit maps (Fig. 4⇓, A and B). In the D86N-Sub structure, OPEP binds to D86N-GzmM without cleavage. The electron density around Ser at the C terminus of OPEP is unclear (Fig. 4⇓A), which might suggest terminal flexibility and weak binding affinity at this site. In the aM-Prod structure, however, no electron density at this site can be observed, indicating the OPEP peptide has indeed been cleaved into CPEP (Fig. 4⇓B).

FIGURE 4.
FIGURE 4.

Determinants of substrate recognition and specificity. Stereo views of D86N-Sub (A) and aM-Prod (B). OPEP/CPEP (green carbon) and substrate binding sites (yellow carbon) are labeled and shown in ball-and-stick representation. 2Fo-Fc omit maps (0.6ς) are colored in gray. Schematic diagram for the interaction between D86N-GzmM and substrate (D86N-Sub) (C) or between active hGzmM and its product (aM-Prod) (D). Initial plots were generated by Ligplot v4.4.2 (43 ) and slightly modified manually. W, water.

The substrate binding sites (S) and the corresponding substrate amino acids (P) for hGzmM could be easily defined from the D86N-Sub and aM-Prod structures. The S1 pocket is located in the catalytic site of GzmM and is composed of Pro177-Cys178, Gly180-Ser182, and Ser199. The S1 site determines the specificity for Leu in the P1 position of the substrate (Fig. 4⇑). Due to the size and hydrophobic properties of the S1 pocket, it can only accommodate long, narrow hydrophobic amino acids such as leucine, methionine, or norleucine, which is consistent with previous reports (15, 37). The S2 pocket is formed by His41, Val80, Leu83, and Phe200, and interacts with the P2 residue of the substrate (Pro) via hydrophobic interactions (Fig. 4⇑). Phe200 and Val83, forming a small valley, limit the space of the S2 pocket and ensure that it can only accommodate Pro or Ala in the P2 position, as reported previously (8, 15). The S3 pocket comprises of the loop Ser201–Arg203 and solvent molecule W4 (Fig. 4⇑). Ser201 and Ser202 form hydrogen bonds with the main chain of the P3 residue (Val) either directly or via W4. The hydrophobic contribution by the side chain of Arg203 is critical for the specificity of the S3 site. The S4 pocket, defined mainly by the groove formed by Pro81, Ala82, Glu84, and Ser160, interacts with the P4 residue (Lys) via hydrogen bonds (Fig. 4⇑). In addition, Leu83 and Phe200 in the S2 pocket supply partial hydrophobic contributions to the interaction with the P4 residue. The calculated electrostatic potential of the S4 pocket is predominantly negative due to the contribution of Glu84. As a result, only residues such as Lys, His, or Arg with a hydrophobic neck and basic head could be accommodated by the S4 pocket. Although the S′ pocket could not be defined clearly in the D86N-Sub or aM-Prod structures, Lys179 is reported to play an important role in substrate recognition (37). It could be involved in formation of the catalytic pocket and might limit the binding specificity at P′ sites.

Conformational change of the L3 loop upon substrate binding

The D86N-Sub structure was superimposed onto the structure of hGzmM (Fig. 5⇓A, left), from which a conformational change of the L3 loop was clearly observed. The most remarkable change occurs in the region of the L3 loop from Phe200 to Arg203 (Fig. 5⇓A, left), while another region of the L3 loop is locked tightly by the disulfide bond DS4 (Figs. 1⇑ and 5⇓A). Although the overall root mean square deviation between the D86N-Sub and hGzmM structures is only 0.35 Å for all Cα atoms, the root mean square deviation for the region of L3 that forms part of the S2 and S3 pockets is ∼2.7 Å. We propose that the movement of these residues should allow the entrance of the optimal substrate into the catalytic site (Fig. 5⇓B). In the substrate-bound D86N-Sub structure, Ser201 switches from the interior to the protein surface, while the phenyl ring of Phe200 changes in the opposite direction, like a lid, to expose the attacking Ser182 residue in the catalytic pocket (Fig. 5⇓A, left and B). Phe200 is thus a pivotal residue to confer substrate specificity of hGzmM, especially for the P2 site where the only preferred residue is Pro or Ala (8, 15). With the exception of the conformational change upon substrate binding, comparing the structures of D86N-Sub and aM-Prod revealed no obvious structural changes as a result of substrate cleavage (Fig. 5⇓A, right).

FIGURE 5.
FIGURE 5.

Conformational change of GzmM upon substrate binding. A, Substrate binding site superpositions between active hGzmM (aM) and D86N-Sub (left panel) or between D86N-Sub and aM-Prod (right panel). aM is colored in orange, D86N-Sub in cyan and aM-Prod in purple. Residues involved in substrate recognition sites and the catalytic triad are shown in ball-and-stick representation. For clarity, only the residues of active hGzmM are labeled. B, Representation of substrate binding pockets of aM (left) and aM-Prod (right) by their electrostatic potential surfaces. The position of Phe200 is indicated. Substrate binding pockets are marked with S1-S4 respectively.

A specific inhibitor of GzmM

Based on structural analysis, the P1 to P4 sites were used to design a peptide inhibitor of hGzmM. The tetrapeptide inhibitor has the sequence KVPL and includes an acetylated N terminus and chloromethylketone (CMK) covalently linked to the C terminus. The CMK group was added to ensure the formation of covalent bonds with both Ser182 and His41 when the inhibitor (Ac-KVPL-CMK) binds to the enzyme, thus irreversibly abolishing the enzyme activity. The crystal structure of hGzmM bound with this inhibitor (aM-Inhibitor) was solved to 2.7 Å resolution. A 2Fo-Fc omit map clearly defined the bound inhibitor in the catalytic site of the enzyme (Fig. 6⇓A). Covalent bonds formed between Leu-CMK and Ser182/His41 effectively lock the inhibitor into the enzyme.

FIGURE 6.
FIGURE 6.

An efficient and specific inhibitor for hGzmM. A, Inhibitor Ac-KVPL-CMK binds to hGzmM at the substrate binding pocket. The inhibitor is shown in ball-and-stick representation with carbon atoms in green; hGzmM is represented by electrostatic surface potential. The inhibitor is cross-linked to Ser182 and His41. 2Fo-Fc omit map (1.2ς) around the inhibitor is displayed as cyan mesh. Negative and positive potentials are colored in red and blue respectively. ACE, acetyl. B, The designed inhibitor Ac-KVPL-CMK can efficiently eliminate the hydrolytic activity of hGzmM by substrate hydrolysis activity assay. I, Inhibitor; E, enzyme. C, GzmM-mediated rTRAP1 cleavage was inhibited by Ac-KVPL-CMK. aM, active GzmM; I/E, inhibitor/enzyme. D, The inhibitor can block GzmM-induced cell death. Jurkat cells treated as described in Materials and Methods were stained with Annexin V and PI, then analyzed by flow cytometry using a FACSCalibur (BD Biosciences). Dead cells were calculated as double Annexin V and PI stained, and shown as mean ± SD% of total analyzed cells. aM, active GzmM; Ad, adenovirus; I, inhibitor. E, The inhibitor cannot inhibit the substrate hydrolysis activity of hGzmA or hGzmB. aA, active hGzmA; aB, active hGzmB; M-I, the inhibitor.

When assaying the proteolytic cleavage activity of hGzmM for a peptide substrate or the protein substrate rTRAP1, the inhibitor was found to block the enzymatic activity of hGzmM in a dose-dependent manner (Fig. 6⇑, B and C). Approximately 90% of the hGzmM enzymatic activity was lost when the molar inhibitor to enzyme ratio (I:E) equalled 1:1. The enzymatic activity was completely lost at an I:E ratio of 4:1. The inhibitor can also abolish hGzmM-induced target cell death when the I:E ratio is larger than 3:1 (Fig. 6⇑D). To further confirm the specificity of this inhibitor among Gzms, we tested its inhibitory activity against GzmA or GzmB. However, no obvious inhibitory effects were observed during the GzmA-mediated rSET degradation or GzmB-induced GST-Bid cleavage experiments at an I:E ratio of 4:1 (Fig. 6⇑E) or even up to 50:1 (data not shown). In summary, the CMK-linked tetrapeptide (Ac-KVPL-CMK) is a specific and efficient inhibitor for human GzmM.

Discussion

From the structure of hGzmM, we observed strong interactions between Asp86 and His41 via a hydrogen bond with a distance of 2.79 Å, and between His41 and Ser182 with a distance of 2.70 Å. The role of Asp in catalytically active Gzm enzymes was therefore re-investigated in this study. Interestingly, we found that a mutation from Asp86 to Asn completely abolished enzyme activity, whereas a mutation from Ser182 to Ala still retained 5–10% of catalytic activity. Similar results were also observed for GzmA and GzmH (unpublished data). These data suggest that classical knowledge derived from the study of trypsin might not be applicable to all serine proteases. In contrast, the acidic Asp86 is indispensable for catalysis, which was further confirmed by pH-dependent enzyme activity assays. Besides the lack of catalytic activity, D86N-GzmM showed very good substrate binding affinity using a BiaCore surface plasmon resonance assay (data not shown) and adopted the same conformation as the wild-type enzyme. This implies that, for Gzm enzymes, a mutant with Asp substituted to Asn would be an ideal catalytically inactive enzyme that could be used as a negative control in functional studies. Alternatively, an Asp-to-Asn mutant could also be used to fish out physiological substrates by affinity chromatography.

In this study, Asp181 was mutated to Asn (D181N-GzmM) to weaken the hydrogen bond between Ile1 and Asp181, resulting in dramatically decreased activity when cleaving the peptide substrate or the protein substrate rTRAP1 (data not shown). This suggests the Ile1-Asp181 hydrogen bond makes an important contribution to the catalytic activity of hGzmM. Any adjustment to the N terminus of hGzmM, such as truncation of the first two Iles (ΔII-GzmM), G3P-GzmM, mutation from Ile1,2 to Leu or to Ala, and adding more residues (SSFGTQ) before the N terminus, would decrease the catalytic activity of hGzmM. Deletion of the first two residues (ΔII-GzmM) or mutation of G3P-GzmM yielded extremely unstable proteins, which might result from the loss of the conformation of the N-terminal insertion. Taken together, these mutation studies suggest that the N-terminal insertion not only aids the stability of the whole structure, but also directly affects the catalytic activity of hGzmM.

Phe200 in the L3 loop is proposed as a major factor in determining the range of GzmM substrates. The flexibility of the L3 loop also enables the possibility that hGzmM might have adaptable substrate specificity and allow the binding of different substrates. This could explain why the preference of the P1 site explored in this study is different to our previously reported cleavage site (Ser in inhibitor of caspase-activated DNase) (14), and why the P2 site is different from the recent report by Cullen et al. (Lys in nucleophosmin) (18).

In this study, we provided one efficient and specific inhibitor for hGzmM and determined its bound structure. This inhibitor (Ac-KVPL-CMK) can completely block the catalytic activity of hGzmM with just a 3- or 4-fold excess in vitro or in cell-loaded assays. Surprisingly, it did not suppress the activities of GzmA or GzmB, even with much more excessive doses. GzmM knockout mice demonstrated increased susceptibility to some viral infections that are reminiscent of those for GzmA- or GzmB-deficient mice (38, 39, 40), indicating the functional redundancy of the Gzm enzymes. Therefore, specific inhibitors are required for the functional investigation of a single member of the Gzm family. GzmM is constitutively and abundantly expressed in innate effector NK cells. Previous reports by Kelly et al. and Lu et al. demonstrated that GzmM-induced rapid cell death is consistent with the kinetics of cytolysis by NK cells (13, 14). NK cells are effectors of innate immunity to trigger adaptive immunity by secretion of IFN-γ, acting as early initiators of immune response (41). Granule-mediated cytotoxicity has been linked to allograft rejection and autoimmune diseases (42). Specific inhibitors to block NK cell-induced cytolysis may, therefore, be a potential approach for organ transplantation or autoimmune diseases. The inhibitory efficiency of the specific inhibitor for GzmM in animal models requires further investigation.

Accession codes

Coordinates of active hGzmM, aM-Prod, D86N-Sub, and aM-Inhibitor have been deposited in the Protein Data Bank (http:// www.rcsb.org) with accession codes 2ZGC, 2ZGH, 2ZGJ, and 2ZKS, respectively.

Acknowledgments

We thank Dr. Yuhui Dong (Beijing Synchrotron Radiation Facility) for help with data collection and processing; Dr. Hongxia Lu for helpful instruction and plasmid construction; Xia Xu and Xudong Zhao of the IBP core facilities centre for technical assistance; Yuanyuan Chen, Peng Xue, and Chunchun Liu for their technical help; and Shuo Wang and Haidong Tang for recombinant protein donations.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported by the National Science Foundation of China (30525005, 30830030, 30623005, and 30772496), 863 program (2006AA02Z4C9, 2006AA02Z173), 973 programs (2006CB504303, 2006CB806506 and 2006CB910901), the Chinese Academy of Sciences (KSCX2-YW-R-42 and the Hundred Talents Program), and the support of K.C. Wong Education Foundation (Hong Kong) to Z.F.

  • ↵2 These authors contributed equally to this work.

  • ↵3 Address correspondence and reprint requests to: Dr. Fei Sun or Dr. Zuse Fan, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China. E-mail address: feisun{at}ibp.ac.cn or fanz{at}moon.ibp.ac.cn

  • ↵4 Abbreviations used in this paper: Gzm, granzyme; aM, active hGzmM; aM-Inhibitor, active GzmM bound to its inhibitor; aM-Prod, active GzmM in complex with its product; CMK, chloromethylketone; CPEP, cleaved peptide; ΔII-Gzm, truncated hGzmM; G3P-GzmM, Gly3 mutated to Pro; hGzm, human Gzm; I:E, molar inhibitor to enzyme; OPEP, octa-peptide; P, substrate amino acid; PI, propidium iodide; S, substrate binding site.

  • Received September 17, 2008.
  • Accepted April 25, 2009.

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