From the Central Research Laboratories and
§ Food Research & Development Laboratories, Ajinomoto
Company Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki,
Kanagawa 210-8681, Japan
Received for publication, October 29, 2000
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ABSTRACT |
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The crystal structure of the tissue-type
transglutaminase from red sea bream liver (fish-derived
transglutaminase, FTG) has been determined at 2.5-Å resolution using
the molecular replacement method, based on the crystal structure of
human blood coagulation factor XIII, which is a transglutaminase
zymogen. The model contains 666 residues of a total of 695 residues,
382 water molecules, and 1 sulfate ion. FTG consists of four domains,
and its overall and active site structures are similar to those of
human factor XIII. However, significant structural differences are
observed in both the acyl donor and acyl acceptor binding sites, which account for the difference in substrate preferences. The active site of
the enzyme is inaccessible to the solvent, because the catalytic
Cys-272 hydrogen-bonds to Tyr-515, which is thought to be displaced
upon acyl donor binding to FTG. It is postulated that the binding of an
inappropriate substrate to FTG would lead to inactivation of the enzyme
because of the formation of a new disulfide bridge between Cys-272 and
the adjacent Cys-333 immediately after the displacement of Tyr-515.
Considering the mutational studies previously reported on the
tissue-type transglutaminases, we propose that Cys-333 and Tyr-515 are
important in strictly controlling the enzymatic activity of
FTG.
Transglutaminase
(TGase1; protein-glutamine
Purification and Crystallization--
Production and
purification of recombinant FTG were performed as previously reported
(29, 30). Crystallization experiments were performed using the method
of vapor diffusion in hanging drops. The crystallization solution in
the reservoir had a volume of 500 ml and was composed of 0.8 M ammonium sulfate, 1% polyethylene glycol 6000, 0.1 M Hepes, pH 7.0, and 5 mM DTT. Two µl of
FTG solution (14 mg/ml) and 2 µl of the reservoir solution were mixed and then equilibrated against the reservoir at 4 °C for 2-4 weeks before crystals appeared.
Data Collection and Processing--
X-ray diffraction data up to
2.5-Å resolution were collected on the beamline BL6B at Photon Factory
(Tsukuba, Japan) using two differently aligned crystals. Because the
crystals suffered from severe radiation damage at room temperature,
they were flash-cooled at Molecular Replacement--
The structure of FTG was solved by
the molecular replacement method, based on the crystal structure of
human factor XIII. The program AMORE (32) was used in the molecular
replacement procedure using intensity data between 10.0 and 3.0 Å for
the rotation search and between 10.0 and 4.5 Å for the translation search. There were single prominent solutions to both the rotation and
translation searches. The solutions indicated that the correct space
group is P6522 and that the asymmetric unit has one FTG molecule, giving a calculated value of ~70% for the solvent content. Rigid body refinement of the orientation and the position of the molecule resulted in a crystallographic R factor of 0.525 at
3.5-Å resolution, and the crystal packing was realistic.
Structure Refinement--
The side chains according to the FTG
sequence were fitted into the initial
2Fo Overall Structure--
The overall structures of FTG and the human
factor XIII monomer are shown in Fig. 1,
and the C
Three cysteine pairs are close enough to each other to form disulfide
bridges. The distances between the Domain Structure--
Both barrel 1 and barrel 2 consist of seven
The
The core domain, in which the active site is located, consists of 11 Structural Differences between FTG and Human Factor XIII--
As
discussed in the previous section, there are some structural
differences in the
The postulated activation process of human factor XIII is as follows.
First, the activation peptide is cleaved by thrombin in the presence of
Ca2+, and then an acyl donor approaches the active site
from the direction of the two barrel domains to form an acyl-enzyme
transition state. Finally an acyl acceptor accesses the active site
from the above-mentioned region to make an
On the other hand, there seem to be few structural differences in the
acyl donor binding site in terms of the C Active Site Structure and Activation Process--
Fig.
6 shows a comparison of the active sites
of FTG and human factor XIII. The catalytic triad reminiscent of the
cysteine proteases consists of Cys-272, His-332, and Asp-355 in FTG and Cys-314, His-373, and Asp-396 in human factor XIII (Fig. 6). Because FTG and human factor XIII have similar active site structures and both
require Ca2+ to exhibit their catalytic activity, their
activation processes are thought to be similar. The catalytic Cys
residue is situated between a Tyr residue (Tyr-515 in FTG and Tyr-560
in human factor XIII) and another Cys residue (Cys-333 in FTG and
Cys-374 in human factor XIII). The Tyr belongs to barrel 1 and is
located on the loop projecting over the core. It seems that O
It has been reported that either the thrombin cleavage or the binding
of one calcium ion alone does not cause the conformational changes of
human factor XIII (23). At present, the activated structure of human
factor XIII has not been elucidated, because substrate binding is
probably required to cause the large conformational changes necessary
for the enzymatic activity.
If the Tyr covering the catalytic Cys were simply removed, then it is
predicted that the Cys would form a disulfide bridge with the adjacent
Cys, and then the enzyme would lose the catalytic activity immediately.
Therefore, we suppose that these two TGases cannot be activated in the
absence of substrates. In other words, the formation of an acyl-enzyme
intermediate is essential to prevent the formation of the disulfide
bridge. There is good evidence to support this hypothesis;
i.e. the catalytic activity of human factor XIII was reduced
to 22% by the proteolysis of the linker peptide joining the core and
barrel 1 domain (28), whereas TGase 3, in which the noncatalytic Cys in
the active site is replaced with Val, was activated 15-fold by the same
treatment (35). We can explain these phenomena by assuming that the
catalytic Cys of human factor XIII formed a disulfide bridge with the
neighboring Cys following the removal of the barrel domains containing
the Tyr covering the catalytic Cys, whereas in the case of TGase 3, the
catalytic Cys was exposed and became more reactive. The remaining activity (22%) of human factor XIII is accounted for by the
presence of DTT in the activity measurement. Consequently, the
activation processes of FTG and human factor XIII are considered to be
as follows. 1) Ca2+ binds to its site in TGase. 2)
An acyl donor is led to the active site. 3) The Tyr covering the
catalytic Cys is removed by a conformational change caused by the acyl
donor binding. 4) An acyl-enzyme intermediate is formed between the
acyl donor and the catalytic Cys (in the case of human factor XIII,
proteolysis of the activation peptide by thrombin is necessary between
steps 1) and 2)). In this activation process, not only the Tyr covering
the catalytic Cys, but also the other Cys near the catalytic Cys, is
considered to play an important role in selecting only appropriate substrates.
Because human factor XIII is the enzyme that forms blood clots, it is
assumed that its activation process is highly controlled to prevent its
activity in the absence of specific substrates, such as fibrin.
Although the physiological role of FTG remains to be determined, its
activation process is also thought to be ingeniously controlled so that
it is activated only under specific conditions. In future studies, the
three-dimensional structure of an FTG-substrate complex should be
determined to demonstrate the activation processes of TGases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-glutamyltransferase) catalyzes the
Ca2+-dependent acyl transfer between the
-carboxyamide groups of glutamine residues within peptides and the
primary amino groups of various amines. A glutamine residue serves as
the acyl donor, and the most common acyl acceptors are the
-amino
groups of peptide-bound lysine residues or the primary amino groups of
some naturally occurring polyamines, like putrescine or spermidine (1).
When a protein-bound lysine residue acts as an acyl acceptor,
intermolecular or intramolecular
-(
-glutamyl)lysine bonds are
formed, resulting in the polymerization of proteins (2-6). TGases are
widely distributed in various organisms (7-18). Human blood
coagulation factor XIII, which forms fibrin clots in hemostasis and
wound healing by catalyzing the cross-linking between fibrin molecules,
has been well characterized (19-28), and its crystal structure has
been determined (22-24). However, the crystal structure is that of an
inactive form, because the catalytic Cys is completely blocked. Human
factor XIII is the enzyme that forms blood clots, and therefore its
activation process is highly controlled so that its catalytic activity
is not exhibited under normal conditions. It is assumed that the structural changes caused by substrate binding as well as the proteolysis of the N terminus by thrombin are necessary to activate human factor XIII (25, 26). Because the activation process is very
complex, the structure-function relationship of human factor XIII
remains unclear, even though the crystal structure has been reported.
To investigate the structure-function relationships of the TGases, the
crystal structures of other TGases will be very helpful. The red sea
bream liver transglutaminase (fish-derived TGase, FTG) is a member of
the tissue-type TGases (29). Although the biological function of FTG
remains unknown, it plays an important role in gel formation in fish
mince sols, by catalyzing a setting reaction that involves the
cross-linking of myosin heavy chains in fish paste. Production and
purification methods for recombinant FTG have already been established
(29, 30), and the amino acid sequence identity between FTG and human
factor XIII is ~33%. FTG has some advantages for studying the
structure-function relationship because FTG works as a monomer, whereas
human factor XIII works as a homodimer, and FTG does not require an
initial proteolysis in its activation process. It is expected that the
activation process of FTG is similar to, but simpler than, that of
human factor XIII. We describe the crystal structure of FTG determined at 2.5-Å resolution and its potential substrate binding sites. We also
propose a novel aspect of the activation process, which involves the
regulation of the enzymatic activity by a Cys residue adjacent to the
catalytic Cys.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
173 °C after equilibration against a
cryo-solvent containing 30% glycerol, 0.6 M ammonium
sulfate, 8% polyethylene glycol 6000, 0.1 M Hepes, pH 7.0, and 5 mM DTT. The diffraction data were processed using
DENZO and SCALEPACK (31). The crystals belong either to space group
P6122 or P6522 with the following unit
cell dimensions: a = b = 97.8 Å; c = 455.5 Å. A summary of the data collection is
shown in Table I.
Summary of the X-ray diffraction data for FTG
Fc map, if
their electron densities appeared clearly. Otherwise, nonconserved
residues were changed to alanine. The model was refined and completed
by rounds of model building using QUANTA97 and simulated annealing
using X-PLOR (33). After the Rcryst dropped to
0.294, water molecules were added to the model. Finally, the
Rcryst and the Rfree
against 37,856 reflections between 8.0- and 2.5-Å resolution were
decreased to 0.196 and 0.246, respectively. The electron densities of
five residues at the N terminus, eleven residues at the C terminus, and
residues 462-471 and 569-571 were not observed. The final model
includes 666 amino acid residues of a total of 695 residues, 382 water
molecules, and 1 sulfate ion derived from the ammonium sulfate used in
the crystallization procedure. The refined structure has two nonproline
cis peptide bonds (Lys-268-Tyr-269 and Lys-384-Tyr-385), as suggested by Weiss et al. (24). The root mean square
deviations in bond length and bond angle are 0.017 Å and 3.454°,
respectively. A Ramachandran plot analysis with PROCHECK (34) reveals
85.7% of residues in most favored regions, 12.6% of residues in
additional allowed regions, 0.5% of residues in generously allowed
regions, and 1.2% of residues in disallowed regions. This result seems quite reasonable for a 2.5-Å resolution model.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
trace of FTG is compared with that of the human factor
XIII monomer in Fig. 2. The sequence alignment of FTG and human factor XIII, together with their secondary structural elements, is shown in Fig. 3.
As shown in Fig. 1, the overall structures of FTG and human factor XIII
resemble each other, although FTG lacks an activation peptide observed
at the N terminus of human factor XIII. FTG, as well as human factor XIII, consists of four sequential domains named "
-sandwich," "core," "barrel 1," and "barrel 2" by Yee et al.
(22). In FTG,
-sandwich contains residues Gly-6 through Phe-134,
core contains residues Asn-135 through Thr-461, barrel 1 contains
residues Arg-472 through Ser-583, and barrel 2 contains residues
Thr-584 through Lys-684. The secondary structures of
-sandwich,
barrel 1, and barrel 2 are predominantly
-sheets, whereas core
consists of almost equal amounts of
-helices and
-sheets
(Fig. 3).
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Fig. 1.
Schematic ribbon drawings of FTG
(left) and human factor XIII
(right). Helices and sheets are colored
red and blue, respectively. Both TGases consist
of four domains: -sandwich, core, barrel 1, and barrel 2 from the N
terminus. Human factor XIII has an activation peptide
(green) at the N terminus. The active site in each TGase is
marked with a yellow asterisk. This figure was produced
using MOLSCRIPT (36).
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Fig. 2.
Structural comparison of FTG with
human factor XIII. Stereoviews of the C traces of FTG and human
factor XIII are shown. The superposition was done using QUANTA97. FTG,
human factor XIII, and the activation peptide are colored
blue, red, and green, respectively.
The active site in each TGase is marked with a white
asterisk.
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Fig. 3.
Structure-based sequence alignment of
FTG and human factor XIII (FXIII). The
dashed lines represent incorporated gaps that bring the
sequences into alignment. The asterisks represent identical
matches. Residues constituting the catalytic triad are colored
red. The red and blue bold lines
represent -helices and
-strands, respectively. The boxes colored
magenta, orange, green, and
blue represent
-sandwich, core, barrel 1, and barrel 2, respectively.
-carbon atoms of the cysteine
pairs are 5.04 Å for Cys-272 and Cys-333, 4.82 Å for Cys-188 and
Cys-285, and 4.82 Å for Cys-523 and Cys-552. Because DTT was added in
the crystallization process, the electron densities between their
sulfur atoms were not observed. However, in the absence of DTT,
disulfide bridges would be formed, except between Cys-272 and Cys-333.
The disulfide bridge formation between the two cysteine residues would
still be prevented by a hydrogen bond between S
of Cys-272 and O
of Tyr-515. The special relationship between Cys-272 and Cys-333 will
be discussed in a subsequent section ("Active Site Structure and
Activation Process").
-strands (Fig. 3). The relative positions of these
-strands
in barrel 1 are quite similar to those in barrel 2 (Fig. 1).
-sandwich domain consists of two four-stranded antiparallel
sheets twisted about 50° with respect to each other. In addition, there is one
-helix in this domain. Although the topologies of the
-sandwich domains of FTG and human factor XIII resemble each other,
the relative positions of the two
-sheets are somewhat different
(Figs. 1 and 2).
-helices and 12
-strands. Most of the
-helices of the enzyme
are located in this domain. As compared with human factor XIII, there
are distinct differences in the
-helix-rich region near
-sandwich
and the region consisting of three
-strands between the active site
and barrel 1.
-sandwich and core domains between FTG and human
factor XIII. Because human factor XIII actually works as a homodimer,
the dimer structure is shown in Fig. 4.
The region between the active site and barrel 1 is adjacent to
-sandwich and the
-helix-rich region of the other half of the
dimer; the activation peptide crosses the interface. This extensive
region is thought to be an acyl acceptor binding site of human factor XIII (22).
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Fig. 4.
Dimer structure of human factor
XIII. Each domain is colored as follows: -sandwich,
red; core, blue; barrel 1, yellow;
barrel 2, orange; and the activation peptide,
green. Possible acyl acceptor binding sites are marked by
magenta circles. This figure was produced using MOLSCRIPT
(36).
-(
-Glu)Lys bond. If
this hypothesis is correct, the structurally different regions between
the two TGases are concentrated on the acyl acceptor binding sites. It
is tempting to postulate that human factor XIII forms a homodimer to
accept only a small subset of proteins as acyl acceptors by increasing the surface area of its binding site.
traces. However, if we
take a closer look at the site, there are distinct differences in the
surface structure and the charge distribution around the acyl donor
binding sites between the two TGases (Fig.
5). In human factor XIII, the acyl donor
binding site forms a deep and wide groove. On the other hand, the
corresponding groove of FTG is shallow and narrow. In addition, there
are some basic regions on the surface of the groove of FTG, whereas the
groove of human factor XIII is scarcely charged. These structural and
electrostatic differences in the substrate binding sites are plausible
causes for the different substrate specificities of FTG and human
factor XIII.
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Fig. 5.
Molecular surfaces of FTG and human
factor XIII. Acidic regions and basic regions are colored
red and blue, respectively. The regions enclosed
by yellow frames represent possible acyl donor binding sites
(22). This figure was produced using GRASP (37).
of the
Tyr and S
of the catalytic Cys form a hydrogen bond, which
suppresses the enzymatic activities of the TGases. Therefore, the Tyr
must be removed to activate the enzymes.
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Fig. 6.
Stereoviews of the active sites of FTG
(A) and human factor XIII (B).
Carbon, oxygen, nitrogen, and sulfur are colored black,
red, blue, and yellow,
respectively.
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ACKNOWLEDGEMENTS |
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We thank Masaru Tanokura for the use of a cryostat and Mamoru Suzuki for help with the initial attempts at data processing with DENZO.
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FOOTNOTES |
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* This study was supported in part by the Sakabe project of the Tsukuba Advanced Research Alliance.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1G0D) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
¶ To whom correspondence should be addressed. Tel.: 81-44-210-5832; E-mail: eiichiro_suzuki@ajinomoto.com.
Published, JBC Papers in Press, November 15, 2000, DOI 10.1074/jbc.M009862200
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ABBREVIATIONS |
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The abbreviations used are: TGase, transglutaminase; FTG, fish-derived TGase; DTT, dithiothreitol.
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REFERENCES |
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