Structural Basis for Proteolytic Specificity of
the Human Apoptosis-Inducing Granzyme M
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J Immunol 2009; 183:421-429; ;
doi: 10.4049/jimmunol.0803088
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References
Lianfeng Wu, Li Wang, Guoqiang Hua, Kan Liu, Xuan
Yang, Yujia Zhai, Mark Bartlam, Fei Sun and Zusen Fan
The Journal of Immunology
Structural Basis for Proteolytic Specificity of the Human
Apoptosis-Inducing Granzyme M1
Lianfeng Wu,*† Li Wang,*† Guoqiang Hua,*† Kan Liu,*† Xuan Yang,*† Yujia Zhai,†
Mark Bartlam,‡ Fei Sun,2,3† and Zusen Fan2,3*†
G
ranzyme (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 (3–7).
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
*Center for Infection and Immunity, and †National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China; and
‡
College of Life Sciences, Nankai University, Tianjin, China
Received for publication September 17, 2008. Accepted for publication April
25, 2009.
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: [email protected] or [email protected]
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; ⌬II-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.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803088
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 ␣-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 –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 wildtype 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 (⌬II-GzmM) were amplified from the full length cDNA of
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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. The Journal of Immunology, 2009, 183: 421– 429.
422
CRYSTAL STRUCTURE OF HUMAN GRANZYME M
Table I. Data collection, phasing, and refinement statisticsa
Data collection
Space group
Unit cell: a, b, c (Å), ␣, ␤, ␥ (°)
Molecule in ASU
Resolution (Å)c
Completeness (%)c
Rmerge (%)c,d
I/␴⬍I⬎b
Refinement statistics
Rwork/Rfree (%)d
r.m.s.d.
Bond length (Å)
Bond angle (°)
Ramachandran plot (%)
Most favored (%)
Allowed (%)
Generous allowed (%)
aM
D86N-Subb
aM-Prod
aM-Inhibitor
P3121
74.4, 74.4, 113.2,
90, 90, 120
1
50-1.96 (2.01-1.96)
98.5 (92.1)
6.5 (34)
28.0 (2.1)
P3121
74.5, 74.5, 112.7,
90, 90, 120
1
50-2.30 (2.36-2.30)
99.4 (99.4)
5.1 (39.9)
36.1 (3.4)
P3121
74.6, 74.6, 112.9,
90, 90, 120
1
50-2.17 (2.25-2.17)
98.9 (98.2)
4.9 (44.8)
20.5 (2.0)
P3121
74.7, 74.7, 113.6,
90, 90, 120
1
57-2.70 (2.77-2.70)
99.8 (99.7)
9.4 (40.8)
7.3 (1.9)
20.9/25.7
24.0/28.8
21.2/25.0
22.3/29.3
0.023
1.726
0.019
1.859
0.017
1.641
0.016
1.880
86.5
13.0
0.5
84.1
15.9
0
85.1
14.4
0.5
80.8
18.7
0.5
a
c
hGzmM (RZPD German Resource Center for Genome Research) by standard PCR cloning strategies. The other mutations were generated by sitedirected 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 ␮g/ml RNase A, 5 ␮g/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 TrisHCl, 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 flashcooling 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,
␭ ⫽ 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.
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 ␮M active GzmM or its variants were incubated with the
substrate (300 ␮M) 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 ␮M rTRAP1 was incubated with 1.5-␮M 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 ␮M 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.
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Corresponding parameters for the highest-resolution shell are shown in parentheses.
D86N-Sub, D86N-GzmM bound to substrate (OPEP).
Rmerge ⫽ ⌺h⌺i兩Iih ⫺ ⬍Ih⬎兩/⌺h⌺i⬍Ih⬎, where ⬍Ih⬎ is the mean of the observations Iih of reflection h.
d
Rwork ⫽ ⌺(㛳Fp(obs)兩 ⫺ 兩Fp(calc)㛳)/⌺兩Fp(obs)兩; Rfree ⫽ R factor for a selected subset (5%) of the reflections that was not included in prior refinement
calculations.
b
The Journal of Immunology
423
FIGURE 1. Stereo view of the active hGzmM structure. ␤-strands (␤1 to
␤13) are labeled and presented in cyan
for domain N and slate blue for domain
C. ␣-helices (␣1 to ␣4) 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.
Results
Overall structure of human GzmM
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 ⴱ 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.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
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 ␣-helices and 13 ␤-strands, is separated into two
domains. The first domain (domain N) is comprised of seven
␤-strands (␤1 to ␤7) and three ␣-helices (␣1, ␣2, and ␣4). The
second domain (domain C) contains six ␤-strands (␤8 to ␤13) and
one ␣-helix (␣3) (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.
424
CRYSTAL STRUCTURE OF HUMAN GRANZYME M
The N-terminal hook is important for GzmM stability and
activity
The catalytic triad and the catalytically inactive mutant
D86N-GzmM
The catalytic triad of hGzmM is composed by Asp86, His41, and
Ser182 (Fig. 2E), 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).
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. 3C), 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
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.
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. 3D)
and reported for GzmH (36).
Despite being catalytically inactive, the structure of the D86NGzmM mutant (data not shown) is identical with that of the wildtype 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
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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. 2A),
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. 2B). The
Gly3 mutated to Pro (G3P-GzmM) mutant and ⌬II-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 I1,2LGzmM (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.
The Journal of Immunology
425
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FIGURE 4. Determinants of substrate recognition and specificity. Stereo views of D86N-Sub (A) and aMProd (B). OPEP/CPEP (green carbon)
and substrate binding sites (yellow
carbon) are labeled and shown in balland-stick representation. 2Fo-Fc omit
maps (0.6␴) 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.
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. 3D). 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.
426
CRYSTAL STRUCTURE OF HUMAN GRANZYME M
Determinants of substrate recognition and substrate specificity
Conformational change of the L3 loop upon substrate binding
The D86N-Sub structure was superimposed onto the structure of
hGzmM (Fig. 5A, 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. 5A, left),
while another region of the L3 loop is locked tightly by the disulfide bond DS4 (Figs. 1 and 5A). Although the overall root mean
square deviation between the D86N-Sub and hGzmM structures is
only 0.35 Å for all C␣ 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.
5B). In the substrate-bound D86N-Sub structure, Ser201 switches
from the interior to the protein surface, while the phenyl ring of
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.
Phe200 changes in the opposite direction, like a lid, to expose the
attacking Ser182 residue in the catalytic pocket (Fig. 5A, 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. 5A, right).
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. 6A). Covalent
bonds formed between Leu-CMK and Ser182/His41 effectively
lock the inhibitor into the enzyme.
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 dosedependent manner (Fig. 6, B and C). Approximately 90% of the
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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. 4A), 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. 4B).
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 D86NSub 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.
The Journal of Immunology
427
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. 6D). 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. 6E) 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 tryp-
sin 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 (⌬II-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 (⌬II-GzmM) or mutation of
G3P-GzmM yielded extremely unstable proteins, which might
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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 (1.2␴) 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.
428
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.
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