(Received for publication, September 15, 1995; and in revised form, December 27, 1995)
From the
We have shown previously that all three fibronectin type II
modules of gelatinase A contribute to its gelatin affinity. In the
present investigation we have studied the structure and module-module
interactions of this gelatin-binding domain by circular dichroism
spectroscopy and differential scanning calorimetry. Comparison of the T values of the thermal transitions of
isolated type II modules with those of bimodular or trimodular proteins
has shown that the second type II module is significantly more stable
in the trimodular protein coll 123 (T
= 54 °C) than in the single-module protein coll 2 (T
= 44 °C) or in the
bimodular proteins coll 23 (T
= 47
°C) and coll 12 (T
= 48
°C). Analysis of the enthalpy changes associated with thermal
unfolding of the second type II module suggests that it is stabilized
by domain-domain interactions in coll 123. We propose that intimate
contacts exist between the three tandem type II units and they form a
single gelatin-binding site. Based on the three-dimensional structures
of homologous metalloproteases and type II modules, a model is proposed
in which the three type II units form an extension of the substrate
binding cleft of gelatinase A.
Proteolytic degradation of constituents of the extracellular matrix and basement membranes plays an important role in tissue restructuring processes such as those accompanying cell migration, morphogenesis, wound healing, angiogenesis, and tumor invasion (Liotta et al., 1991). The proteinases implicated in tumor invasion include components of the urokinase receptor/urokinase/plasminogen system and several members of the metalloproteinase family (He et al., 1989; Matrisian, 1992; Vassalli and Pepper, 1994). Enzymes capable of degrading type IV collagen are crucial for tumor metastasis as this collagen is a major component of basement membranes which have to be penetrated during migration of tumor cells. Significantly, secretion of type IV collagenases is well correlated with metastasis and transformation (Librach et al., 1991; Stetler-Stevenson et al., 1993).
Studies on the primary structures of 72-kDa and 92-kDa type IV collagenases (gelatinase A and B) have revealed that they contain a catalytic domain, a hemopexin-like domain, and three tandem homology units closely related to the type II domains of fibronectin (Collier et al., 1988; Wilhelm et al., 1989).
The hemopexin-like domain of gelatinase A has been shown to be involved in modulation of its activity by TIMPs, the tissue inhibitors of metalloproteases (Murphy et al., 1992; Fridman et al., 1992). This region is also required for the activation of progelatinase A by a membrane-associated activator, suggesting that it may interact with some component of the cell membrane (Murphy et al., 1992; Vassalli and Pepper, 1994).
Studies on the substrate specificity of gelatinases have shown that they are able to degrade type IV, type V, type VII, and type X collagens, fibronectin, elastin, and all types of denatured collagens (Murphy et al., 1991; Senior et al., 1991; Matrisian, 1992).
Gelatinases are unique among metalloproteinases in having pronounced affinity for denatured collagens. Recently it was shown that the gelatin-binding sites of gelatinases reside in their fibronectin-related regions. Recombinant proteins corresponding to the fibronectin-related regions of gelatinase A and B were found to have high affinity for gelatin (Bányai and Patthy, 1991; Collier et al., 1992; Bányai et al., 1994). Conversely, recombinant gelatinase A lacking the fibronectin type II units was shown to be devoid of affinity for gelatin or type I and type IV collagens, indicating that the fibronectin-like domain is the sole site of collagen binding (Murphy et al., 1994; Allan et al., 1995).
The functional significance of the gelatin-binding site is suggested by the observation that although deletion of this domain does not affect the catalytic properties of gelatinase A on small synthetic substrates (Ye et al., 1995), activity on gelatin is drastically reduced and the cleavage pattern of type IV collagen is altered (Murphy et al., 1994; Ye et al., 1995). On the basis of these observations, it has been proposed that the fibronectin-like domain of gelatinase A specifically orientates the enzyme on type I gelatin or type IV collagen, thus enhancing the rate of substrate cleavage (Murphy et al., 1994).
To clarify the role of the collagen-binding domain, we have initiated studies to define its interaction with substrates. In a previous paper (Bányai et al., 1994), we described the expression of type II domains of the fibronectin-related region of gelatinase A in Escherichia coli and the characterization of their type I gelatin-binding properties. We have shown that although each of the three type II domains binds gelatin, proteins containing all three tandem type II domains of gelatinase A have significantly higher affinity than any of the constituent units: all three type II units contribute to gelatin binding. In the present work, we have studied the collagen-binding domain by circular dichroism spectroscopy and differential scanning calorimetry to elucidate the structure and interactions of the three type II modules.
Reduced-alkylated DELgalcoll 123 was prepared as follows:
DEL
galcoll 123 (0.025 mM) was dissolved in 0.1 M Tris-HCl, 6 M guanidinium HCl, 5 mM EDTA, 50
mM dithiothreitol, pH 8.0, and the solution was incubated at
25 °C for 30 min. The reduced-denatured protein was alkylated with
iodoacetic acid (110 mM). The alkylated protein was desalted
by gel filtration on a Sephadex G-25 column equilibrated with 0.1 M ammonium bicarbonate, pH 8.0, and the protein was lyophilized. For
some experiments, reduction-alkylation of DEL
galcoll 123 was
carried out in a similar way, except that guanidinium HCl was omitted
from the reaction mixture.
The concentration of the recombinant proteins was determined spectrophotometrically using extinction coefficients determined according to a described method (Mach et al., 1992). Protein samples were analyzed by SDS-polyacrylamide gel electrophoresis using 11-22% or 16-22% linear polyacrylamide gradient slab gels (Laemmli, 1970). The gels were stained with Coomassie Brilliant Blue G-250.
Unfolding of recombinant proteins containing the fibronectin type II domains of gelatinase A was monitored by the changes in their CD spectra at 224 nm. To monitor thermal unfolding of type II units, circular dichroism was recorded at 224 nm during the course of heating the solutions from 15 °C to 90 °C at a heating rate of 50 °C/h. The experiments were conducted at pH 8.0, 10 mM Tris-HCl, the protein concentration was 0.5-1.0 mg/ml. Melting temperatures were determined by derivative processing of the CD changes using the J-700 program for Windows Standard Analysis Ver.1.30.00.7, JASCO.
The far UV CD spectra of DELgalcoll 123 (containing the
entire fibronectin-related part of human gelatinase A), as well as the
CD spectra of recombinant proteins containing single type II modules
are characterized by maxima at 224 nm and minima at 198 nm (Fig. 1). Removal of the
-galactosidase fusion peptides had
no major effect on the CD spectra of these proteins (cf.
DEL
galcoll 123 and coll 123 in Fig. 1). Analysis of the CD
spectra has shown that the fibronectin-related domain of gelatinase A
consists of about 32%
-sheet, 19%
-turn with no detectable
helix. Recombinant proteins coll 1, coll 2, and coll 3 were
estimated to contain 30%, 30%, and 31%
-sheet, 28%, 27%, and 23%
-turn structures, respectively.
Figure 1:
Circular
dichroism spectra of DELgalcoll 3 (-),
DEL
galcoll 123(- - -), coll 123
(
), and reduced-alkylated DEL
galcoll123
(-
-). Spectra were obtained in 10 mM Tris-HCl, pH 8.0 at 25 °C.
The estimated secondary
structures of type II domains of gelatinase A are in good agreement
with the predictions based on their homology with the second type II
domain of PDC-109. The solution structure of the latter protein has
been solved by NMR spectroscopy (Constantine et al., 1992),
and these studies have revealed the presence of two short antiparallel
-sheets connected by
-turns, with no evidence for
helix. Secondary structure predictions by the method of Chou and
Fasman(1978) also suggested the presence of
-turns and
-sheets but no
helix in fibronectin type II domains (Patthy et al., 1984). This conclusion was confirmed and extended in
the present work for type II modules of gelatinase A and B as well as
PDC-109 (Esch et al., 1983), BSP A3 (Seidah et al.,
1987), mannose 6-phosphate receptor (Morgan et al., 1987),
blood coagulation factor XII (McMullen and Fujikawa, 1985), hepatocyte
growth factor activator (Miyazawa et al., 1993), mannose
receptor (Taylor et al., 1990), and phospholipase receptor
(Ishizaki et al., 1994) using the multiple alignment-based
method of Rost and Sander(1994). In agreement with our earlier findings
(Patthy et al., 1984), the type II modules of these proteins
are predicted to have four short
-strands in the vicinity of their
four half-cystines and an additional
-strand is predicted in the
N-terminal extension that is present only in some of the type II units (Fig. 2). It is noteworthy that the predicted
-strands
align with the four
-strands determined experimentally for PDC-109
domain 2. The CD spectra of type II modules are similar to those of the
related kringle domains inasmuch as they also have characteristic
maxima near 225 nm (Castellino et al., 1986). In accordance
with the homology of kringles and type II domains (Patthy et
al., 1984), both are characterized by the presence of antiparallel
-sheets and
-turns (Constantine et al., 1992).
Figure 2:
Alignment and secondary structure of the
fibronectin type II units of gelatinase A (GA1, GA2, GA3), gelatinase B (GB1, GB2, GB3),
fibronectin (FN1, FN2), mannose receptor (ManR), phospholipase A2 receptor (PLR), blood
coagulation factor XII (FXII), hepatocyte growth factor
activator (HGFA), mannose 6-phosphate receptor (Man6PR), bovine seminal fluid protein A3 (A31, A32), and PDC-109 (PDC1, PDC2). The bottom line shows the secondary structure predicted by the PHD
program (Rost and Sander, 1994): capitals denote positions
with an expected accuracy higher than 82%; E, -strand; L, not helix or
-strand. In the case of the second type
II domain of PDC-109 (PDC2) for which the solution structure
has been determined by NMR spectroscopy (Constantine et al.,
1992), the segments known to form
-sheets are underlined.
The maxima at 224 nm are characteristic of the ordered, native
structure of the type II modules of the gelatin-binding domain. For
example, denaturation of DELgalcoll 123 eliminates this maximum (Fig. 1); the reduced-alkylated protein has a minimum at 200 nm,
typical of unordered proteins (Chang et al., 1978). The
disulfide bonds of type II units are essential for the integrity of
their three-dimensional structure since reductive cleavage of S-S bonds
of DEL
galcoll 123 in the absence of denaturing agents leads to a
CD spectrum characteristic of the fully denatured form and eliminates
its affinity for gelatin-Sepharose 4B (not shown). (It must be pointed
out that this finding contrasts with the conclusion of Steffensen et al.(1995); these authors suggested that reductive cleavage
of the disulfide bonds has no effect on the functional integrity of
these domains).
Unfolding of the recombinant proteins with urea or
guanidinium HCl eliminates the maxima at 224 nm in a highly cooperative
fashion. As shown in Fig. 3, the sensitivity of DELgalcoll
1, DEL
galcoll 2, and DEL
galcoll 3 to denaturant-induced
unfolding is markedly different: the midpoints of unfolding with
guanidinium hydrochloride are 0.3 M, 1.6 M, and 4.8 M for DEL
galcoll 2, DEL
galcoll 1, and
DEL
galcoll 3, respectively. The order of sensitivity of type II
domains to urea-induced unfolding is similar: the midpoints of
unfolding with this denaturant are 1.7 M and 6.5 M for DEL
galcoll 2 and DEL
galcoll 1, respectively. The
most stable DEL
galcoll 3 is not unfolded even in 8 M urea.
Figure 3:
Unfolding of DELgalcoll 1 (*),
DEL
galcoll 2 (
), DEL
galcoll 3 (
), and
DEL
galcoll 123 (
) with guanidine HCl (A) and urea (B). Changes in the CD of the proteins were monitored at 224
nm in 10 mM Tris-HCl buffer, pH 8.0 at 25
°C.
It may be pointed out that unfolding of DELgalcoll
123 with guanidinium HCl occurs in three steps, the midpoints of these
steps coincide with unfolding of the individual type II units (Fig. 3A). In contrast with this, unfolding of
DEL
galcoll 123 with urea is not detectable below 3 M urea, even though unfolding of DEL
galcoll 2 is practically
complete at this concentration (Fig. 3B). It seems
likely that interactions in DEL
galcoll 123 increase the stability
of the second type II domain to urea-induced unfolding.
Thermal
unfolding was also monitored by changes in the CD spectra of type II
modules at 224 nm. These studies have shown that the three type II
modules show marked differences in their thermal stability ( Fig. 4and Table 1). The least stable is the second type II
module with a T value of 44 °C, the most
stable is the third unit with a T
of 80 °C.
Differences in their stability are retained in the recombinant proteins
that contain two or three type II domains, permitting the resolution of
the thermal transitions into distinct components (cf. Fig. 4and Table 1). From a comparison of recombinant
proteins containing the N-terminal fusion peptide (DEL
galcoll
series) with those lacking this segment (coll series), it is also clear
that the N-terminal peptide does not have a major effect on the thermal
transition of type II modules (Table 1).
Figure 4:
Temperature dependence of the circular
dichroism of DELgalcoll 1(- - -),
DEL
galcoll 2 (
), DEL
galcoll 3
(-
-), DEL
galcoll 13 (-
-), and DEL
galcoll 123 (-). Changes in the CD
of the proteins were monitored at 224 nm in 10 mM Tris-HCl
buffer, pH 8.0, during the course of heating from 15 °C to 90
°C at a heating rate of 50 °C/h. Melting temperatures were
determined by derivative processing using the J-700 program for Windows
Standard Analysis Ver.1.30.00.7, JASCO.
When the T values of the second type II module of different
recombinant proteins are compared (Table 1), it is clear that
neighboring modules have a marked influence on its thermal stability.
This is most obvious in the comparison of the T
value (44 °C) of type II unit 2 of coll 2 with its T
value (54 °C) in full-length
collagen-binding domain (coll 123): the presence of module 1 and module
3 increases its T
value by 10 °C. The presence
of module 1 or module 3 alone (in coll 12 or coll 23) causes a smaller
but significant increase in thermal stability of module 2 (Table 1). It thus seems likely that the second type II domain
interacts tightly with both module 1 and module 3.
Differential
scanning calorimetry studies (e.g.Fig. 5) yielded T values similar to those obtained by monitoring
changes in CD spectra (Table 1). The melting curves of the
individual domains are each well described by single two-state
transitions, consistent with their
H
/
H
ratios close
to unity (data not shown). Comparison of the enthalpy changes of the
unfolding of type II domain 2 in coll 123 (
H
= 58 kcal/mol) and coll 2 (
H
= 49 kcal/mol) indicates that domain-domain interactions
in coll 123 significantly stabilize the folded state of domain 2.
Figure 5: Differential scanning calorimetry of coll 3.
In the present investigation we have shown by analysis of the
CD spectra of recombinant proteins that the type II domains of the
gelatin-binding site of type IV collagenase have a protein-fold
characterized by a high content of -structures, with no
-helix. This observation is in harmony with structural information
obtained on a related type II domain, the second unit of the bovine
seminal fluid protein PDC-109 (Constantine et al., 1992).
Secondary structure prediction from multiple alignments of type II
units also supports the presence of
-structures but no
-helix
(Patthy et al.(1984) and the present study).
CD
spectroscopy and differential scanning microcalorimetry of the
collagen-binding domain has shown that the thermal stability of the
second type II module is significantly increased by its interactions
with the first and third modules: its T value is
increased from 44 °C to 54 °C. The fact that the stability of
the second type II domain to urea-induced unfolding is increased by the
presence of the other two type II units also suggests that these units
are involved in tight interactions. It seems likely that these
interactions between the tandem modules permit little flexibility in
their relative orientation at physiological temperatures. In view of
the fact that all three type II units contribute to gelatin binding
(Bányai et al., 1994), we propose that
their fixed arrangement facilitates the tight binding of a single
substrate molecule.
The binding of the substrate by the type II domains may play a crucial role in the proper positioning of these substrates relative to the active site cleft of gelatinase A. Although the three-dimensional structure of gelatinase A has not yet been determined, structures of proteins homologous to its three-constituent domains: the catalytic domain (Lovejoy et al., 1994a, 1994b; Borkakoti et al., 1994; Gooley et al., 1994; Stams et al., 1994; Bode et al., 1994), hemopexin-like domain (Faber et al., 1995; Li et al., 1995), and fibronectin type II domain (Constantine et al., 1992) have been determined recently.
The catalytic domains of other members of
the matrixin family of metzincins MMP-1, MMP-3, and MMP-8 (Lovejoy et al., 1994a, 1994b; Borkakoti et al., 1994; Gooley et al., 1994; Stams et al., 1994; Bode et
al., 1994; Stöcker and Bode, 1995) have been
shown to have very similar three-dimensional structures. They all
comprise a regularly folded ``upper'' subdomain consisting of
a twisted five-stranded -sheet flanked by two
helices and
connecting loops, and a smaller, less regularly folded,
``lower'' subdomain comprising two open loops and the
C-terminal helix. The catalytic zinc ion residing at the bottom of the
active-site cleft between these subdomains is bound by a characteristic
helix-bend-loop structure comprising the
HEXXHXXGXXH consensus sequence. The
catalytic zinc ion is coordinated by the imidazolyl N
2 atoms of
the three consensus histidine residues and by a water molecule that is
also bound to a glutamate (Bode et al., 1994;
Stöcker and Bode, 1995).
On the basis of the
close homology of gelatinase A (MMP-2) with other matrix
metalloproteinases, it is safe to assume that its three-dimensional
structure is also similar to these enzymes, except that three
fibronectin-related type II units are inserted at the N-terminal
boundary of the active site helix of gelatinase A, seven residues
upstream of the HEXXHXXGXXH consensus sequence motif.
Homology modeling of the catalytic domain of gelatinase A indicates
that the type II domains are inserted at the right-hand end of the
active-site cleft, close to the S` pocket of the enzyme (Fig. 6). In view of the proximity of the collagen-binding type
II units to the active site cleft, it is conceivable that they are
crucial for properly orienting the substrate relative to the catalytic
site, presenting the scissile bond to the active site of the enzyme.
Such a role of the collagen-binding domain is supported by the
observation that the cleavage pattern of type IV collagen is altered in
a mutant gelatinase A lacking this domain (Murphy et al.,
1994). In accordance with the experimental data, we suggest a model in
which the three type II units form an extension of the substrate
binding cleft of gelatinase A (Fig. 6).
Figure 6: Structural model of gelatinase A. The upper figure shows the exploded view of the catalytic and fibronectin-related domains of gelatinase A. Homology modeling of the individual domains was performed with the Swiss-Model Automated Protein Modelling Service using known structures of related metzincins and the second fibronectin type II domain of PDC-109. Note that the fibronectin-related region of gelatinase A is inserted at the right-hand corner of the upper lip of the active-site cleft (white segment). The three histidines coordinating the catalytic zinc ion are shown in red. The lower figure shows a hypothetical model of gelatinase A consistent with the experimental data. In this model, the three type II units form a hydrophobic groove lined by the aromatic residues (purple) that are known to be involved in gelatin binding in the case of the type II domain of PDC-109 (Constantine et al., 1992). This groove may serve to bind and orient the substrate relative to the catalytic site, presenting the scissile bond to the active site of the enzyme. The figures were prepared with Insight II (Biosym Techn, San Diego).