 |
INTRODUCTION |
The biological importance of factor XIII
(fXIII)1 (EC 2.3.2.13) lies
in its ability to form new covalent bonds between protein chains. This
activity was first recognized while studying blood coagulation; fXIII
was required to form an insoluble clot (1). The activated form of fXIII
covalently cross-links two fibrin molecules via an isopeptide bond
between the side chains of a glutamine and a lysine located in the
C-terminal region of the
-chain. Over a longer time period in
coagulation, it also forms cross-links between the
- and
-chains
of fibrin (2), and between
2-anti-plasmin and fibrin
(3). fXIII has been shown to react with more than fibrin (4), and it
has recently been found in brain tumors (5) and arthritic joints (6),
and there are cases of fXIII deficiencies (7, 8). Factor XIII is a
member in the family of transglutaminases (TGases), which have a wide
range of biological functions (9).
Like most coagulation factors, fXIII is synthesized as a zymogen and
then cleaved by a protease to become an active enzyme. The structure of
fXIII zymogen was determined several years ago (10, 11). In this
crystal form, the active site cysteine, Cys-314, is inaccessible to
solvent and is not available for catalysis.
Physiologically, calcium ions are required for fXIII activation and for
TGase activity. In the blood, activation of circulating fXIII requires
thrombin cleavage, calcium ions (1.5 mM) (12-14), and
fibrin(ogen) (15). High levels of calcium (>50 mM) can
activate fXIII without the use of thrombin (15), and it has recently been shown that platelet fXIII can be activated nonproteolytically in vivo (16).
Based on the amino acid sequence, the calcium binding site was
predicted to be in a region (residues 468-479) with high similarity to
the EF-hand motif (17, 18). The main calcium binding site, as seen in
the preliminary crystallographic study (19), involves residues Asn-436,
Asp-438, Ala-457, Glu-485, and Glu-490. C-terminal truncation
experiments removing several residues from the calcium binding pocket
show a total loss of enzymatic activity (20). Site-directed mutagenesis
of the glutamate residues in the calcium binding region of guinea pig
liver TGase decreases its sensitivity to calcium, while maintaining
some enzymatic activity (21).
There is further in vitro evidence that calcium and other
divalent cations have effects on proteolytic susceptibility (22) and
heat stability (23). Several lanthanide ions have been shown to replace
calcium ions during fXIII activation (10-40 µM).
Furthermore, these lanthanide ions can inhibit fXIII activity when the
ion concentration is greater than 40 µM (24). Most of
these effects occur when the calcium ion is in the millimolar range,
and with the lanthanide ion in the micromolar range.
We have solved the structures of recombinant fXIII zymogen complexed
with calcium, strontium, and ytterbium to determine the structural
effects of these cations on fXIII. The goal in using the latter two
cations was to positively identify the electron-rich ions in the
electron density maps. All three ions bind to the same pocket. We have
found an additional ytterbium binding site, hypothesized to be the
lanthanide inhibition site.
 |
EXPERIMENTAL PROCEDURES |
Crystallization and Data Collection--
Crystals of recombinant
human factor XIII A2 (166 kDa) (25) zymogen were grown from
1, 2-propanediol (24%) and sodium potassium phosphate buffer (100 mM) at pH 6.2. The crystals show the symmetry of space
group P21 with two monomers in the asymmetric unit. The ion-bound crystals were obtained by soaking zymogen crystals for 1-2
days in 90 mM CaCl2, 120 mM
SrCl2, or 2 mM YbCl3 in an
artificial mother liquor of 1,2-propanediol (24%) and MES buffer at pH
6.2. Data for the calcium soaked crystal were collected at the Stanford Synchrotron Radiation Laboratory on beamline 9-1 with a MAR image plate detector at
170 °C. For the strontium and ytterbium
crystals, data were collected in-house at room temperature on an R-AXIS IIc image plate detector equipped with a Rigaku RU200 rotating anode
generator. The calcium crystal yielded data to 2.1 Å, and the
strontium and ytterbium data sets diffracted to 2.5 Å (Table I).
Structure Refinement--
Our previously refined P21
fXIII zymogen structural model was used to generate initial phases for
all three ion-bound structures. The initial R (
||Fobs|
|Fcalc||/
|Fobs|) was 48.2%, 36.8%, and 31.0% for
the calcium, strontium, and ytterbium structures, respectively.
The calcium refinement began with a series of rigid body minimization
steps at 8-3.5 Å resolution using X-PLOR (26). The first rigid body
refinement used two monomers, while the next was composed of the eight
domains; finally, a refinement with 193 secondary structure elements
(helix, strand, loop) was performed. After those three steps, the
R had dropped from 48.2% to 32.9%. This multistep rigid
body procedure was used because of the cryogenic data collection and
higher initial R factor. This rendered a 2.3% lower
R factor than if a simple two-monomer rigid body step was used. From here, the resolution was expanded to 2.2 Å in six steps by
alternating rigid body (with 193 groups) and positional least square
refinements. The R dropped to 27.9% with an
Rfree of 38.6%. Simulated annealing with slow
cooling from 2000 to 300 K, followed by a round of positional and
B value least squares minimization gave a drop in
R of 0.4% and Rfree of 1.6%.
Refinement continued through several rounds of map fitting with the
program XtalView (27) and least squares and dynamics minimization. The
program DDQ (28) was used to assess model and map quality during the refinement procedure. Waters were added at positions with difference electron density peaks greater than 3.0
, which also were within hydrogen bonding distance of the protein or other waters. In the end,
the structure was refined with overall anisotropic scaling of the data
along with a bulk solvent model and a resolution of 20.0-2.1 Å; this
resulted in an R of 22.7% and Rfree
of 31.2%, with a total of 1001 waters (Table I).
The strontium and ytterbium refinements were carried out over the
resolution range of 10-2.5 Å for all steps. The X-PLOR refinement began with a positional and individual B value minimization
of the starting model, which lowered the R to approximately
27%, and was followed by adding two ions per dimer at the highest
peaks in the difference map (Table II). It then proceeded through more cycles of least squares minimization and manual map fitting in the
program O (29). In the ytterbium refinement, three additional 10-12
difference peaks were observed near the non-crystallographic two-fold axis within the dimer, and three ions were added (sites 3, 4, and 5). Furthermore, one additional Yb3+ ion was added at a
10
difference peak (site 7), which is located at a crystallographic
contact with another dimer. Following refinement of these four ions in
the structure, one more difference peak appeared on the two-fold axis,
so another ytterbium ion was added (site 6). After further refinement
and map fitting, the R decreased to 19.8% (strontium) and
22.0% (ytterbium), and waters were placed at chemically reasonable 3
difference peaks. Additionally, a strong difference peak about 2 Å away from the main Yb3+ (site 2) in monomer B was
identified, and a new Yb3+ ion (site 8) was added with an
occupancy of 0.25, while the occupancy of the original Yb3+
ion was reduced to 0.75. Several more rounds of least squares refinement, map fitting, and water addition led to a final R
of 18.3% and 18.8%, an Rfree of 27.5% and
28.1%, and 230 and 265 waters for the strontium and ytterbium
complexes, respectively (Table I).
View this table:
[in this window]
[in a new window]
|
Table I
Data collection and refinement statistics
NA, not available; SSRL, Stanford Synchrotron Radiation Laboratory.
|
|
The Protein Data Bank codes for the calcium, strontium, and ytterbium
structures are 1GGU, 1BL2, and 1GGY, respectively. Figures in this
report were generated with Molscript (30), Raster3D (31), and XtalView
(27).
 |
RESULTS |
Overall Structure--
Each monomer of fXIII in the dimer has 731 residues and consists of four domains: the
-sandwich, the catalytic
core, and barrels 1 and 2 (Fig. 1). The
active site residues Cys-314, His-373, and Asp-396 are not accessible
to solvent, as in the zymogen structure. The calcium binding site is
located in the core domain, near the surface of the protein. The root
mean square deviation (r.m.s.d.) between all atoms from the strontium-
and ytterbium-bound structures compared with the starting ion-free
fXIII zymogen structure is less than 0.85 Å. The calcium
structure, on the other hand, differs from the two cation structures
and the starting structure with an r.m.s.d. of 1.1 Å. This
higher atomic coordinate deviation is likely due to the cryogenic data
collection for the calcium structure.

View larger version (47K):
[in this window]
[in a new window]
|
Fig. 1.
Overall structure of the factor XIII
dimer. Panel A is the dimer looking down the
two-fold axis. The domains, activation peptide (AP), and N
and C termini of one monomer are labeled. Panel B
shows one monomer, rotated 90° with the domains, N terminus, active
site (AS), and ion sites labeled. For both views, three
unique spheres are also shown: novel ytterbium site on the dimer
two-fold axis (medium gray), active site
(light gray), and main ion site (dark
gray).
|
|
Due to uninterpretable electron density, the monomers are missing
various numbers of residues from the N terminus (residues 1-8), the
linker between the activation peptide and the
-sandwich (residues
30-43), the region between the core and barrel 1 (residues 508-516),
and the C terminus (residues 728-731). Most of the residues were well
resolved, with 90% having average B values of less than 60 Å2.
Structure Quality--
Less than 0.4% of the torsion angles are
in the forbidden regions of the Ramachandran diagram, as reported by
PROCHECK (32). Three peptide bonds in each monomer are in the
cis conformation (10, 33). One involves a proline residue,
410-411, and the other two do not, 310-311 and 425-426. The
structures are reasonable as judged by DDQ (28), Verify3D (34), ERRAT
(35), and WHATIF (36).
Main Ion Site--
The main ion binding site is near the interface
between the catalytic core and barrel 1 (Fig. 1), and the ion binding
helix (residues 485-501) is in contact with the other monomer. The
calcium and strontium atoms superimpose within this site, while the
main ytterbium ion is 2.7 Å from their location (Fig.
2). The mean peak sizes in an early
difference map were about 4, 7, and 11
for the calcium, strontium,
and ytterbium ions, respectively (Table
II). When water molecules replaced the
calcium ions at these positions, their B values refined to
about 14-23 Å2. However, for the strontium and ytterbium
sites, the B values of the replaced water molecules refine
down to 2 Å2, the minimum allowable value. This analysis
implies that a water molecule in this location does not have enough
electrons to adequately match the strontium and ytterbium x-ray
diffraction data.

View larger version (57K):
[in this window]
[in a new window]
|
Fig. 2.
The main calcium binding site. The
calcium (red), strontium (yellow), ytterbium
(blue), and zymogen (gray) structures from
monomer B are superimposed in this figure. Both positions of the
Yb3+ ion are shown, with the weaker ion represented by the
smaller sphere and labeled with
parentheses. The water molecules in this region have been
omitted for simplicity.
|
|
The main cation site consists of a pocket with negatively charged side
chains of Asn-436, Asp-438, Glu-485, and Glu-490 plus the main chain
oxygen of Ala-457. All three ion-bound structures and the zymogen
structure overlap quite closely in this region (Fig. 2). Table
III lists the oxygen atoms that are
potentially involved in coordinating each cation. The main chain oxygen
from Ala-457 is the main protein ligand for the Ca2+ and
Sr2+ ions, as indicated by its low B value and
its distance from the metals. The Yb3+ ion is farther from
Ala-457 and closer to Glu-485 and Glu-490. The B values for
the residues in the region from 484 to 490 are higher than average, and
the electron density is weak.
The ion site in monomer B of the calcium structure has six putative
ligands arranged in a distorted bipyramidal arrangement (Fig.
3). One protein atom (O-Ala-457) and up
to five waters are within 4.0 Å of the ion. Residues Asp-438, Asn-436,
and Glu-490 are hydrogen-bonded to several of these waters. Fewer
waters were identified in the strontium and ytterbium structures due to
the lower resolution data.

View larger version (89K):
[in this window]
[in a new window]
|
Fig. 3.
Stereoview of the electron density for the
calcium site. Shown is the final 2|Fo| |Fc| electron density (1.2 contour)
for the calcium binding site in monomer B of the calcium structure. The
residues are labeled with the single-letter code
for amino acid and residue number. Waters from
Table III are labeled, and all waters are shown as crosses.
Two waters (300L and 23L) have visible electron density at a lower
contour level (0.5 ; not shown).
|
|
The ytterbium ion has one monodentate (Asn-436) and two bidentate
(Glu-485, Glu-490) ligands provided by the protein and nearby water
molecules. The ion site in monomer B of the ytterbium structure has two
alternate positions for the Yb3+ ion 1.8 Å from each
other. The occupancies for each ion were chosen such that they refined
to equal B values and summed to 1. It is unlikely that the
weaker Yb3+ ion (site 8) is a negative chloride ion because
it is very close to the position of the positive calcium ion from the
calcium structure, which implies that its location is suitable for only
positive ions. The ligand distances between the Yb3+ ion
and the oxygen atoms are as low as 1.98 Å, but this is consistent with
other Yb3+-oxygen distances, as observed in the Protein
Data Bank (codes 1CNT (37), 1NCG and 1NCH (38), 1YTT (39), and 2BOP
(40)).
Novel Ytterbium Site--
Four large electron density peaks near
the non-crystallographic dimer two-fold axis form a nearly perfect
tetrahedron with an edge length of approximately 3.6 ± 0.1 Å (Fig. 4 and Table II). Furthermore, a
similar tetrahedron is observed in the ytterbium soak of the
orthorhombic crystal form (10) of fXIII (data not shown). After
modeling four ytterbium ions at 0.25 occupancy into the electron
density, the B values refined to 17, 23, 23, and 10 Å2. Placing waters at full occupancy in these locations
gave B values that refined to 2 Å2. No ions or
solvent molecules exist in the same location in the Ca2+-
or Sr2+-bound crystal structures. Residues Asp-270,
Asp-271, and Glu-272 of each monomer are in this region, but the
electron density does not specify one distinct conformation for the
side chains, and the B values are quite high.

View larger version (82K):
[in this window]
[in a new window]
|
Fig. 4.
Stereoview of the electron density for the
novel ytterbium binding site. Shown is the final
2|Fo| |Fc|
electron density (1.0 and 5.0 contour levels) for the novel
ytterbium site. This tetramer of ions is located on the dimer two-fold
non-crystallographic axis. The residues are labeled with the
single-letter code for amino acid and
residue number. The top residues are from
monomer A, and the bottom residues are from monomer B.
|
|
Water Analysis--
After the refinement of the three structures,
waters were grouped into subsets based on their occurrence in each
crystal. If waters from different structures were located within 2.4 Å from each other, they were considered equivalent, and given the same
chain identification and residue number in the Protein Data Bank files.
In some cases, the determination of equivalent waters was made on the
basis of their B values and local protein differences between structures. A Venn diagram was constructed (Fig.
5) showing the seven different subsets
from the three independent groups. If a water from one structure had no
equivalent waters from another structure, it was given a particular
chain identifier (L for calcium, R for strontium, Y for ytterbium). If
a water molecule was in only two different structures, it was given the
same residue number and chain identification (U for calcium and
strontium, V for calcium and ytterbium, W for strontium and ytterbium).
Finally, water molecules that exist in all three structures were given
the chain identification of S along with the same residue number.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 5.
Venn diagram of the resulting subsets of
water molecules from the three ion structures. Shown above are the
total number of waters and their assigned chain identification for each
subset. Each water is placed in only one of the possible seven
subsets.
|
|
The average and standard deviation of B values and
C
density for the water molecules are presented in Table
IV. The C
density (41)
estimates the amount of burial at each water site by counting the
number of C
atoms within 10 Å of the water site. It has
a numerical range of 2-30 atoms, which qualitatively ranges from
highly exposed to deeply buried.
View this table:
[in this window]
[in a new window]
|
Table IV
Water subsets: average B values and C densities
Average C density is the average of the number of
C atoms within 10 Å of each water in the subset. PDB,
Protein Data Bank; ID, identification.
|
|
Many of the water molecules are located in sites common to all three
structures. Water molecules that are present in all three structures
(subset S) have B values about 10 Å2 lower than
unique waters. Likewise, these common waters are more buried, as shown
by the 50% larger number of nearby C
atoms.
 |
DISCUSSION |
Overall Structure--
Calcium and other cations have definite
effects on fXIII behavior in solution. The ion soaking concentrations
in the calcium and strontium crystals were above the level at which
conformational changes, such as enzyme activation, are expected. No
significant conformational differences between the ion complex and the
zymogen structure are seen. Two complementary reasons for this lack of conformational change are: 1) the active conformation of the enzyme cannot pack in this crystal form, and 2) some other molecule, such as a
substrate or inhibitor, is required to stabilize fXIII in its active form.
Packing interactions in the monoclinic crystal form restrict any major
conformational changes induced in fXIII. Crystallization of thrombin
cleaved fXIII in the presence of 1.5 mM calcium has resulted in the same crystal form and the same conformation (42). Furthermore, co-crystallization of 45 mM CaCl2
with fXIII has yielded this monoclinic crystal form with the same
conformation (43). Structurally, one residue that must move to expose
the active site Cys-314 is Tyr-560, located in barrel 1. In this space group, barrel 1 is trapped in place by crystal contacts. We therefore believe that this crystal form is incompatible with the active form of fXIII.
The biochemical experiments used to demonstrate the effects of calcium
and other cations on fXIII use either a substrate or inhibitor to
measure whether the enzyme is active. We propose that one of these
molecules is another requirement for the stabilization of the active
form. In the presence of calcium, the inactive form of fXIII is in
equilibrium with the active conformation. During crystallization, the
inactive form is more likely to be crystallized; thus, mass action
causes any active fXIII to change to its inactive conformation. The
presence of a substrate or inhibitor might stabilize the active
conformation further and allow crystallization in a new crystal form.
This proposal is in line with the known effect of fibrin(ogen) in
facilitating the activation of fXIII (14, 44).
Main Ion Site--
Some ideas about calcium activation can be
gained from the location of the main binding site with respect to other
features on the molecule. First, the calcium binding pocket is only 10 Å from barrel 1, which is responsible for partially blocking the active site via Tyr-560. The bound ion may induce some dynamical changes in nearby barrel 1, including exposure of the active site. Additionally, one end of the calcium binding helix forms close contacts
with the other monomer. This may allow for allosteric communication
between the calcium binding site of one monomer and the active site of
the other, as described by Hornyak and Shafer (14).
The residues involved in calcium binding are well conserved within the
TGase family. The coordination geometry for the calcium and strontium
is not ideal; many of the putative ligands are water molecules, and the
metal-ligand distances are somewhat longer than observed for calcium
complexes in other proteins (45) (Table III). The B values
for these residues are not much different from those observed for the
ion free structures. These observations are consistent with fXIII's
millimolar binding affinity (15) for Ca2+ ions. The
Yb3+ ion, which binds more tightly, is in a slightly
different location and has more coordinating protein atoms with fewer
water ligands. This ion has a smaller radius and has a higher charge
than divalent cations, so the fact that it has a different binding
location is not unreasonable.
Novel Ytterbium Site--
The four ytterbium ions on the dimer
two-fold axis are modeled with 1/4 occupancy each. The
interatomic distances are too small to allow multiple positively
charged ions to be present concurrently. They are time and space
averaged throughout the crystal so that only one ion is in the site at
any given time. This multiplicity of the ion has made it difficult to
model the protein ligands.
We postulate that this tetrahedron of partial occupancy ions is the
lanthanide inhibition site. Achyuthan et al. (24) have observed that the addition of lanthanide ions above 40 µM
to thrombin-cleaved fXIII results in the non-competitive inhibition of
fXIII. This inhibition cannot be reversed by a 200-fold molar excess of
calcium ions. Furthermore, when the level of the lanthanide ion is
between 10 and 40 µ M, fXIII can be activated by thrombin
cleavage as in the presence of calcium. These results (24) could be
explained by multiple lanthanide binding sites. The main ion site,
observed in all three ion-bound structures, is required for proper
thrombin activation, and the novel Yb3+ site, which has a
slightly weaker affinity, is responsible for the inhibition of fXIII.
Given the soaking concentration of 2 mM in this data set,
both ions sites are filled. Functionally, the location at the dimer
interface could potentially destabilize the quaternary structure.
Additionally, the residues that form the lanthanide inhibition site
(Asp-270 to Glu-272) are only 5 residues away from Trp-279, which may
play an important role in catalysis (11).
Waters--
Grouping the waters into subsets based on their
presence in multiple structures has been helpful in identifying
structurally significant waters. One example is that of water 6059S
(Fig. 6). This water in the core domain
binds to several residues that are fully conserved in the TGase family
(Thr-466, Lys-467, Arg-333, and Tyr-204). The water is located at the
separation fork of a pair of
strands. One half of each strand forms
a paired sheet, and the other half of each strand diverges nearly at a
right angle. Furthermore, these strands are linked with functionally
important parts of the protein. One strand pairs with the strand
containing the active site His-373; and the other strand contains a
ligand (Ala-457) for the calcium binding site. Monomer B also contains a water molecule in this location (6009S). We believe this water is
structurally important for maintaining the unique conformation of this
pair of strands.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 6.
Structurally significant water molecule.
Water 6059S is shown as a sphere with several secondary structure
elements nearby. The residues near this water are also shown. The
active site residue His-373 and calcium binding ligand Ala-457 are
shown.
|
|
Many other waters in this subset S are contacting conserved residues in
this protein, and it is possible that some of these waters may help to
explain natural fXIII deficiency mutations or other interesting
structural and evolutionary features. In fact, of the 18 identified
missense mutations (46-49)2
that cause a fXIII deficiency, half are contacting at least one conserved water from subset S. The probability of this happening by
chance is 5%.
Conclusion--
There are no major conformational changes between
the different ion-bound fXIII structures and the zymogen. This is
likely due to crystal packing interactions and the lack of a substrate or inhibitor molecule to stabilize the active conformation. The location of the main ion binding site provides ideas about the mechanism of calcium activation, such as its proximity to barrel 1 and
its contact with the other monomer. The novel ytterbium binding site,
on the dimer two-fold axis, could be responsible for the biochemically
observed lanthanide inhibition. The procedure of finding common waters
between the structures is a new method for identifying particular
waters and residues that may be structurally significant and important
for the function of fXIII. Overall, these three structures positively
identify the calcium binding site in factor XIII.