From the Faculty of Dentistry and the Department of
Biochemistry and Molecular Biology, Faculty of Medicine, University of
British Columbia, Vancouver, British Columbia V6T 1Z3, Canada and the
¶ Division of Hematology-Oncology, Children's Hospital Los
Angeles and University of Southern California,
Los Angeles, California 90027
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ABSTRACT |
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Cell surface activation of progelatinase A occurs
in a quaternary complex with the tissue inhibitor of
metalloproteinases-2 (TIMP-2) and two membrane-type matrix
metalloproteinases. We have mutated the unique cationic clusters found
in hemopexin modules III and IV of the carboxyl domain (C domain) of
human gelatinase A to determine their role in binding TIMP-2. Twelve
single, double, and triple site-directed mutations were produced that
exhibited different TIMP-2 binding properties. Notably, single alanine
substitutions at Lys547 and Lys617
reduced TIMP-2 binding by an order of magnitude from that of the
recombinant wild-type C domain. Mutations that completely disrupted the
C domain·TIMP-2 interaction were K558A/R561A, K610T/K617A, and
K566A/K568A/K617A. A triple mutation, K566A/K568A/K575A, having TIMP-2
binding indistinguishable from the wild-type C domain
(Kd 3.0 × 10 Connective tissue remodeling is important for growth, healing, and
functional adaptation of tissues. In these processes, activation of
matrix metalloproteinase
(MMP)1 zymogens is a key
control step in the degradation of extracellular matrix proteins
(reviewed in Refs. 1 and 2). Gelatinase A (EC 3.4.24.24) (MMP-2) is a
pivotal MMP in the remodeling of basement membrane, pericellular, and
cell attachment proteins. Cellular activation (3, 4) and other cell
membrane binding properties (5, 6) of gelatinase A are central to the
regulation and function of this enzyme (reviewed in Ref. 7). The tissue inhibitors of metalloproteinases (TIMPs) form essentially irreversible 1:1 molar inhibitory complexes with active MMPs (8, 9) and so also
control MMP activity. Inhibition includes critical interactions between
the MMP catalytic Zn2+ ion and Cys1 of the TIMP
inhibitory NH2-domain (10, 11). However, outside the active
site TIMP-2 (12, 13),
TIMP-3,2 and TIMP-4 (14) also
bind progelatinase A in a 1:1 molar complex on the hemopexin
carboxyl-terminal domain (C domain) of the enzyme. That is, active
gelatinase A can simultaneously bind two molecules of TIMP-2: one at
the active site, to inhibit the enzyme, and another on the C domain.
Similarly, TIMP-1 binds the progelatinase B C domain (15), but two
molecules of TIMP-1 cannot bind gelatinase A (16).
We initially demonstrated that Con A induced the endogenous cellular
activation of progelatinase A (3). Paradoxically, the progelatinase A C
domain and TIMP-2 carboxyl domain interaction appears essential for
enzyme activation on the cell membrane in this process (4, 17, 18).
Upon binding TIMP-2, a trimolecular complex of progelatinase A first
forms with an activator proteinase, membrane type (MT)-MMP (18-20). In
this interaction, the ihibitory NH2 domain of TIMP-2 binds
and inhibits the MT-MMP active site (20-23). We (24) also have
proposed that a quaternary activation complex then forms with a second
MT-MMP that cleaves the prodomain of progelatinase A at
Asn37-Leu38. The domain binding interactions of
these proteins and their roles in the activation complex are not yet
fully resolved. However, deletions and use of isolated domains have
shown that TIMP-2 binds progelatinase A via the C domain of both the
enzyme (14, 16, 17, 20, 25, 26) and the inhibitor (26,
27).3 Nonetheless, the
localization of the respective contact surfaces on these two protein
domains and the important molecular determinants of the binding sites
have yet to be identified. In this regard, Willenbrock et
al. (26) proposed that the highly charged anionic peptide
extension (186QEFLDIEDP194) present at the
carboxyl terminus of TIMP-2, but not of TIMP-1, mediates binding to the
gelatinase A C domain. Of note, TIMP-4 also contains a similar sequence
(187KEFVDIVQP195) (28) and binds to the
gelatinase A C domain (14). Thus, these carboxyl-terminal tails may be
crucial in forming the gelatinase A binding site on these TIMPs. In
addition, an alternate interaction of the gelatinase A C domain with
The overall shape of the gelatinase A C domain is a squat cylinder
composed of four In the absence of a three-dimensional structure of the
TIMP-2·gelatinase A complex, we adopted a mutagenesis approach to
identify the TIMP-2 binding site on the gelatinase A C domain. Rather
than individually mutate all 36 basic residues in the C domain, we devised a strategy of using multiple replacements to screen more efficiently for important amino acid sites. Further, these
substitutions were only made in the third and fourth hemopexin modules,
which contain the unique cationic clusters not found in other MMPs Sequence and Three-dimensional Modeling Analyses--
To devise
a rational mutagenesis strategy, we first utilized the Clustal V method
(35) for multiple sequence alignments of MMP C domains to locate unique
and conserved cationic residues in the human gelatinase A C domain. The
strategy was later refined using structural models of the domain. Until
the three-dimensional structure was reported (30, 31), we used a
molecular model that was made with ProMod to predict the tertiary fold
and CHARMM for energy minimization. As a template, the coordinates of
the porcine collagenase-1 hemopexin domain (36) were used; they were
most kindly provided by Dr P. Brick (Blackett Laboratory, Imperial
College, London, United Kingdom) before publication.
Mutagenesis--
Using the method of Kunkel et al.
(37), 12 site-directed mutations of the human gelatinase A C domain
(Gly417-Cys631) were made, and the mutant
cDNAs were fully sequenced by double strand dideoxy chain
termination. As listed in Table I, the
following sites were mutated as indicated: K547A, K549A, K550A, K575A,
K617A, K558A/R561A, K566A/K568A, K550A/K566A/K568A, K566A/K568A/K575A, K566A/K568A/K617A, K547A/K549A/K550A, and K610T/K617A. Amino acid numbering commences at the NH2 terminus of the proenzyme.
Although an alanine substitution was planned for Lys610,
the mutagenesis reaction was not efficient and produced nucleotide changes in only a single clone that coded for Thr610. The
location of the sites mutated on the upper face of the C domain are
depicted in a three-dimensional structural model in Fig.
1.
Recombinant Protein Preparation--
Recombinant human TIMP-2
was expressed in Chinese hamster ovary cells and purified from the
conditioned medium as described previously (38). Recombinant human
gelatinase A C domain and mutant proteins were expressed with an
NH2-terminal His6 tag (39) in 60-liter
fermenter cultures of Escherichia coli strain Le392 using a
100-liter L & H fermenter. During culture, the temperature was
controlled at 37 °C, airflow was 7.5 liters/min, air saturation was
controlled at 5%, and mixer agitation was 400 rpm (39). After 18 or
42 h of culture, recombinant protein was purified from the
harvested cells (~310 g) essentially according to Steffensen et
al. (40) using procedures that have been characterized as generating correctly folded and biologically functional recombinant C
domain (14, 34). We have previously reported the essential role of the
structural Ca2+ ion in stabilization of the C domain (34).
Therefore, to further optimize the refolding environment, 5 mM CaCl2 was included in the 0.1 M
Na2B4O7, pH 10, refolding buffer.
The purity of the wild-type and mutant domain proteins was assessed by
SDS-polyacrylamide gel electrophoresis and Western blotting using two
affinity purified antipeptide antibodies: Binding Assays--
Binding of the C domain and mutant proteins
to TIMP-2 was measured using a solid phase microwell plate assay (14)
with bovine serum albumin and myoglobin as negative control proteins.
TIMP-2 was coated onto 96-well plates at 0.2 µg per well in 15 mM Na2CO3, 35 mM
NaHCO3, 0.02% NaN3, pH 9.6, for 18 h at
4 °C. Wells were then blocked with 2.5% bovine serum albumin, 0.1%
NaN3 in PBS for 1 h at 21 °C. Wild-type and mutant
C domain proteins were serially diluted and added to the TIMP-2 coated
wells under physiological conditions in 100 µl of PBS for 2 h at
21 °C. Unbound protein was removed by extensive washing with PBS,
0.05% Tween 20 using an automated plate washer. Binding of C domain or
mutant proteins to TIMP-2 was measured using affinity purified
anti-peptide Affinity Chromatography--
The NH2-terminal
His6 fusion tag was utilized to bind wild-type and mutant C
domains with high affinity to Zn2+- or
Ni2+-charged chelating-Sepharose 6B resin (Amersham
Pharmacia Biotech) in 75-µl minicolumns made from pipette tips (41).
Following extensive washes in PBS to remove unbound protein, a
>10-fold molar excess of TIMP-2 in PBS was applied to the column,
which was then washed with PBS, 0.05% Brij 35. TIMP-2 elution was
attempted with 1.0 M NaCl, then 10% Me2SO, and
finally 50 mM EDTA. Elutes were electrophoresed on 15%
SDS-polyacrylamide gels and stained with Coomassie Brilliant Blue R250
to detect TIMP-2 in the column fractions. Relative amounts of the
EDTA-eluted C domain (or mutant proteins as appropriate) and TIMP-2
were quantitated by laser densitometry of the protein bands. Affinity
chromatography was performed at least three times per protein.
To test the hypothesis that cationic residues in the gelatinase A
C domain bind TIMP-2, possibly at the anionic terminal tail, multiple
sequence alignment analysis was first used to devise a rational
mutagenesis strategy. As shown in Fig. 2,
15 of the 26 basic residues in the C domain are grouped in hemopexin
modules III and IV. Comparing the distribution of cationic residues in the C domains of gelatinases A and B, there are more nonconserved basic
residues in modules III and IV than in I and II. Therefore, we
predicted that the cationic sequences in hemopexin modules III and IV
were more likely to be involved in binding TIMP-2. We first focused on
the markedly cationic module III, which contains 10 basic residues
compared with 5 in module IV, and designed mutations to disrupt the
lysine-rich sequences Lys547-Asn-Lys-Lys550 and
Lys566-Lys-Lys568. Three point mutations were
made to assess the TIMP-2 binding properties of the individual lysines
in the Lys547-Asn-Lys-Lys550 sequence. A double
mutation (K566A/K568A) was also used to test the effect of charge
reduction in the Lys566-Lys-Lys568 triad.
8 M),
showed that simple reduction of net positive charge does not reduce
TIMP-2 affinity. Because the double mutation K566A/K568A also did not
alter TIMP-2 binding, these data do not confirm previously reported
chimera studies that indicated the importance of the triple lysine
cluster at positions 566/567/568 in TIMP-2 binding. Nonetheless, a
subtle role in TIMP-2 interaction for the 566/567/568-lysine triad is
indicated from the enhanced reduction in TIMP-2 binding that occurs
when mutations here were combined with K617A. Thus, these analyses
indicate that the TIMP-2 binding surface lies at the junction of
hemopexin modules III and IV on the peripheral rim of the gelatinase A
C domain. This location implies that considerable molecular movement of
the TIMP-2·C domain complex would be needed for the bound TIMP-2 to
inhibit in cis the gelatinase A active site.
INTRODUCTION
Top
Abstract
Introduction
References
v
3 integrin has been described (5) but
not confirmed (22), and a lower affinity interaction also occurs
between the NH2-domain of TIMP-2, but not that of TIMP-1 or
TIMP-3, and the gelatinase A C domain (24, 27).3 Finally,
the MMP-independent growth factor effects of TIMP-2 (29) have been
ascribed to the C domain of the inhibitor. These may be masked upon
binding the gelatinase A C domain. Thus, locating the TIMP-2 binding
site on the gelatinase A C domain and generating mutations that disrupt
this interaction will be invaluable in dissecting the mechanistic
aspects and relative importance of the different cell membrane binding
and activation mechanisms proposed for progelatinase A. These
mutant proteins will also be useful in assessing the consequences of
TIMP-2 binding to the gelatinase A C domain on the growth factor
properties ascribed to TIMP-2.
-sheets, each representing a hemopexin module
(I-IV) and each forming a blade of the four bladed
-propeller structure (30, 31). Each
-sheet is formed from four antiparallel
-strands. Analysis of these three-dimensional models has revealed cationic clusters in hemopexin module III as a striking feature. These
largely result from contiguous stretches of lysine residues, Lys547-Asn-Lys-Lys550 and
Lys566-Lys-Lys568, located near the
antiparallel
-strand turns at the rim and on the upper surface of
the domain. Notably, Lys550, Lys566, and
Lys567 from these sequences also contribute to three
consecutive BX7B repeats (B = basic
residue, X = undefined):
[Lys550-Lys558],
[Lys558-Lys566], and
[Lys567-Lys575]. A fourth
BX7B motif
[Lys617-Lys625] is found in module IV.
BX7B motifs have been implicated in hyaluronan binding in several proteins (32). Although the gelatinase A C domain
binds heparin (33, 34), which enhances progelatinase A activation (22,
33), it does not bind
hyaluronan.4 However, these
cationic clusters and BX7B repeats may be
important in binding TIMP-2, possibly interacting through salt bridge
formation (24). This hypothesis has been tested in the present report.
in particular gelatinase B, which binds TIMP-1 but not TIMP-2. We reasoned
that multiple substitutions should also lessen the risk of missing
weaker binding residues that as single mutations may have their effects
masked by the overall binding energy of the TIMP-2·C domain complex.
Reported here are mutations that establish an important role in TIMP-2
binding for several basic residues in the gelatinase A C domain.
Analysis of the effects of these mutations and their location on the
three-dimensional structure of the C domain has also enabled us to
define the approximate boundary of the TIMP-2 binding site. Thus,
TIMP-2 binds the upper surface of the human gelatinase A C domain, on
its outer rim at the junction of hemopexin modules III and IV. This
location has a number of ramifications concerning the proposed actions
of the bound TIMP-2.
EXPERIMENTAL PROCEDURES
Mutation sites in the human gelatinase A C domain
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Fig. 1.
Three-dimensional model of the upper surface
of the human gelatinase A C domain showing the amino acid sites
substituted by site-directed mutagenesis and the mutant proteins
generated. The spacefill graphic of the C domain was made using
coordinates deposited in the Protein Data Bank by Gohlke et
al. (31) (Protein Data Bank accession number 1RTG). The top of the
pseudosymmetry axis with the central Ca2+ ion on the upper
face of the domain is shown. The location of hemopexin modules I to IV
is indicated (HxI-HxIV). All lysine and arginine residues
are colored black. Not visible in this view are
Lys575 and Lys558, which are under the domain
near Arg561. The general location of these sites is
indicated by the jagged arrow labeled with the mutations
involving Lys575.
His6 (34) and
72Ex12. Anti-72Ex12 antiserum was raised in rabbits against a
KLH-coupled peptide from the sequence
561RYNEVKKKMDPG572 in the gelatinase C domain
that encompasses the lysine triad at positions 566/567/568. To confirm
the predicted molecular mass and homogeneity of the recombinant
wild-type and mutant proteins, electrospray mass spectrometry was
performed on a PESCIEX API 300 as before (14). Protein concentrations
were calculated using the molar extinction coefficient (5.10 × 104 M
1 cm
1) of the
recombinant C domain determined previously from quantitative amino acid
composition analysis (14). The Lys
Ala, Lys
Thr, and Arg
Ala mutant proteins were assumed to have the same extinction coefficients as the wild-type recombinant C domain. The purified recombinant proteins were stored in phosphate-buffered saline (PBS)
(140 mM NaCl, 2.7 mM KCl, 4.3 mM
Na2HPO4·7H2O, 1.5 mM
KH2PO4, pH 7.4), 0.05% Brij 35 at
70 °C
after snap freezing in liquid N2.
His6 antibody followed by incubation with
goat anti-rabbit alkaline phosphatase conjugated secondary antibody
(Bio-Rad). After adding p-nitrophenyl phosphate disodium
(Sigma) as substrate, absorbance measurements were made at 405 nm in
the linear range of the assay using a Thermomax plate reader (Molecular
Devices). Binding constants were determined by curve fitting using
SigmaPlot on a Macintosh computer.
RESULTS
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Fig. 2.
Multiple sequence alignment of MMP hemopexin
C domains. The sequences of the hemopexin domains shown are from
human MMPs, except for MMP-18, for which only the Xenopus
sequence is currently available. The amino acid sequences shown are
from the first cysteine residue involved in the single intradomain
disulfide cross-linkage. The linker from the COOH-terminal Cys to the
transmembrane helix of the MT-MMPs is not shown. The hatched
pattern indicates a gap in the numbering. The location of
hemopexin (Hx) modules I-IV are as indicated. Second
line, the amino acid sequence of the hemopexin domain of human
gelatinase A (MMP-2) is shown with residue numbers commencing from the
processed amino-terminal amino acid residue of the secreted
progelatinase A protein (lacking the signal peptide). Sites mutated in
the gelatinase A C domain are indicated by underlining.
Conserved residues of other MMP C domains are indicated by
dots, gaps by hyphens. GenBankTM accession
numbers for the sequences used are as follows: MMP-1, M13509; MMP-2,
P08253; MMP-3, X05232; MMP-7, X07819; MMP-8, J05556; MMP-9, J05070;
MMP-10, X07820; MMP-11, X57766; MMP-12, P39900; MMP-13, P45452; MMP-14,
P50281; MMP-15, P51511; MMP-16, P51512; MMP-17, X89576; MMP-18, L76275;
MMP-19, Y08622; and MMP-20, Y12779.
Fermenter conditions for each mutant protein were optimized. Expression
conditions were mutant protein-specific, but in general, oxygen-limited
conditions (5% aeration) proved optimal for the expression of the
recombinant gelatinase A C domain and mutants thereof. The mass (Da)
measured by electrospray mass spectrometry and the mass from that
predicted for the wild-type C domain protein and from the mass of the
first round of mutant proteins expressed is shown in Table
II. These data confirmed the homogeneity of the protein preparations, that NH2-terminal methionine
processing occurred in the recombinant proteins, and that the correct
amino acid substitution had been translated.
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Mutagenic Analysis of the TIMP-2 Binding Properties of the
Lys547, Lys549, and Lys550
Cluster--
A differential role in TIMP-2 binding was observed for
alanine-substituted proteins at positions 547, 549, and 550 on the upper surface of hemopexin module III (Table
III). An alanine mutation at
Lys547 raised the apparent Kd of TIMP-2
interaction by approximately 1 order of magnitude (apparent
Kd 1.7 × 107 M) and
reduced the amount of TIMP-2 bound by ~50% at saturation (Fig.
3A). Mutation of
Lys549 or Lys550 to alanine did not
significantly alter the apparent Kd values of TIMP-2
interaction (7.8 × 10
8 M (Fig.
3A) and 4.8 × 10
8 M (Fig.
3B), respectively) from that of the wild-type domain (3.0 × 10
8 M). TIMP-2 binding and
elution from these mutant proteins on affinity chromatography columns
was also no different from the wild-type C domain (not shown). Because
a cooperative effect on TIMP-2 binding by residues in this cluster was
possible, we then made the triple alanine mutation (K547A/K549A/K550A).
However, TIMP-2 binding by the K547A/K549A/K550A protein (apparent
Kd, 2.6 × 10
7 M) was
essentially no different from that of the K547A mutant (Fig.
3A). This indicated that cooperative binding by these three residues does not occur and that of these residues, Lys547
plays the most important binding role.
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The enigmatic role of
Lys566-Lys567-Lys568 in TIMP-2
Binding--
A striking feature of the C domain sequence is the unique
cationic triad Lys566-Lys-Lys568 on hemopexin
module III (see Figs. 1 and 2). This triad also contributes to two
BX7B motifs:
[Lys558-Lys566] and
[Lys567-Lys575] (see Table I). Rather than
initially mutate each of the three residues separately, we first
screened for the TIMP-2 binding properties of this cluster as a whole
by charge reduction using a double alanine mutation. Sites 566 and 568 were selected, as these were nearer than Lys567 to the
other cationic residues in module III (see Fig. 1). The mutant
K566A/K568A protein showed TIMP-2 binding properties (apparent Kd 4.7 × 108 M)
(Fig. 3C) that were essentially identical to those of the wild-type C domain. Moreover, an affinity purified antibody raised against a peptide (NH2-RYNEVKKKMDPG-COOH) encompassing the
lysine triad did not interfere with TIMP-2 binding to the wild-type C domain immobilized on a Zn2+-chelate affinity column (Fig.
4A). Finally, chromatography
of TIMP-2 over a K566A/K568A protein affinity column revealed strong TIMP-2 binding to the mutant domain. Like the wild-type C domain (Fig.
4A), this interaction was not disrupted by 1.0 M
NaCl or 10% Me2SO
the bound TIMP-2 and K566A/K568A mutant
domain complex were eluted in 50 mM EDTA (not shown). We
have previously shown that fibronectin and heparin binding by the
gelatinase A C domain is Ca2+ ion-dependent,
being disrupted by divalent cation chelators (34). In microwell plate
assays, the C domain was not dissociated from TIMP-2 by 50 mM EDTA (not shown). This indicates that the EDTA elution
of C domain with TIMP-2 from the affinity columns was primarily due to
chelation and removal of the ligating Zn2+ ion from the
metal chelate resin. Thus, the C domain and TIMP-2 in the 50 mM EDTA eluates represent the bound complex. Because the
K566A/K568A double mutation did not alter TIMP-2 binding, these two
residues were not individually substituted nor was Lys567
mutated.
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Of the basic residues in the
Lys547-Asn-Lys-Lys550 sequence,
Lys550 is topographically closest to the
Lys566-Lys-Lys568 triad (see Fig. 1).
Therefore, to determine the effects of simultaneous mutations in these
two sequences, we made the triple mutation K550A/K566A/K568A. In
addition, this selection would also disrupt both the
[Lys550-Lys558] and
[Lys558-Lys566] BX7B
repeats (Table I). In this regard, Lys550, rather than
Lys558, was also the more rational selection because
Lys558 is on the underside of the domain. We considered
this surface a less likely TIMP-2-binding region candidate given that
7 of the 10 basic residues in hemopexin module III are on the upper surface. As already discussed, the K550A and K566A/K568A mutations alone showed little alteration in apparent Kd values for TIMP-2 compared with the wild-type C domain. However, as a triple
mutation (K550A/K566A/K568A), a reduction in binding by approximately 1 order of magnitude resulted (apparent Kd, 1.8 × 107 M) (Fig. 3B). This
indicates that Lys550, and possibly
Lys566/Lys568, forms contacts with TIMP-2.
However, unlike Lys547, these appear weak and contribute
little to the overall binding energy of the complex.
Mutation of Basic Residues at Positions 558, 561, and 575--
The
four antiparallel -strands in the
-sheet of module III place
Lys558 and Lys575 on the lower surface of the
domain. Lys575 lies between Lys558 and
Arg561, which is located on top of the domain on the outer
rim near Lys550 (see Fig. 1). Neither the single point
mutation K575A (apparent Kd, 5.2 × 10
8 M) nor the triple mutation
K566A/K568A/K575A (apparent Kd, 7.5 × 10
8 M) significantly perturbed the TIMP-2
interaction. Indeed, these two mutant proteins showed very similar
binding properties to that of the wild-type C domain both in the plate
assay (Fig. 3D) and by affinity chromatography (not shown).
However, the mutation K558A/R561A showed almost total loss of TIMP-2
binding (Fig. 3D). Therefore, it is likely that in the
K558A/R561A double mutant protein, Arg561 has the major
TIMP-2 binding role of this pair.
The Importance of Lys617 in Module IV for TIMP-2
Binding--
All the mutations described in the preceding sections
were in module III. To investigate the contribution to TIMP-2 binding by basic residues in module IV, three mutant proteins were made. Lys617 is on the outer rim of this module near the upper
surface of the domain (see Fig. 1). The mutant protein K617A bound to
TIMP-2 in a saturatable manner but with reduced affinity, as determined from the reduction in apparent Kd by nearly 1 order
of magnitude (1.1 × 107 M) (Fig.
3C). This result indicates an important role in the TIMP-2
interaction for Lys617 and that TIMP-2 also contacts
hemopexin module IV. As already discussed, the mutation K566A/K568A had
little effect on TIMP-2 binding. However, when this double mutation was
combined with K617A, a synergistic decrease in TIMP-2 affinity resulted
that reduced binding to undetectable levels (Fig. 3C).
Consistent with this, TIMP-2 did not bind to K566A/K568A/K617A mutant
protein affinity columns, with all the applied TIMP-2 being collected in the unbound fractions (Fig. 4B). In contrast, TIMP-2
bound to the K617A mutant protein and could be eluted with this protein in 50 mM EDTA (Fig. 4C). However, the relative
amount of TIMP-2 bound to the mutant protein and recovered in the
eluates was quantitated at ~0.5-fold that of the amount of TIMP-2
bound to the wild-type C domain. The plate assays were consistent with
this finding. The curve fitting analysis confirmed a near 50%
reduction in the total amount of K617A protein bound to TIMP-2 at
saturation compared with the wild-type C domain.
The importance of position 617 and module IV was further shown by the
K610T/K617A mutation. This reduced TIMP-2 binding to undetectable
levels both in the plate assay (Fig. 3B) and by affinity chromatography (Fig. 4B). Thus, Lys610 would
also appear to play an important role in TIMP-2 binding, but a
confirmatory point mutation of this site is needed for an unequivocal demonstration.
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DISCUSSION |
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Salt bridge formation between the unique cationic clusters in the
hemopexin-like C domain of progelatinase A and the anionic carboxyl-terminal peptide tail of TIMP-2 is predicted to be important for complex formation between the gelatinase A C domain and TIMP-2. This hypothesis was tested by generating 12 single, double, and triple
site-directed mutations of 10 basic residues in the human gelatinase A
C domain in order to locate the TIMP-2 binding site. Our studies have
shown that disruption of TIMP-2 binding to the C domain follows the
introduction of an energetically unfavorable change in side chain
polarity at several sites, in particular at Lys547 in
hemopexin module III and at Lys617 in module IV. Two double
mutants also strongly implicate Arg561 and
Lys610 as playing an important role in TIMP-2 binding.
Finally, analysis of several triple mutations indicates that a weak
TIMP-2 interaction occurs with Lys550 and reveals a subtle
role in TIMP-2 binding for Lys566 and Lys568.
Our data do not reveal a canonical linear sequence that forms the
TIMP-2 binding site, nor do they show a critical role in TIMP-2 binding
for the four BX7B motifs present in hemopexin
modules III and IV of the domain. Only two of seven residues covered by these motifs are implicated in TIMP-2 binding, and they are not one of
the BX7B pairs. Rather, these repeats, in
combination with the periodicity of the turn of the antiparallel
-strands, serve to position lysines near to each other in the
tertiary structure of the domain. This concentration of charged
residues forms cationic clusters on the upper surface of the
domain
some residues in each, but not all, we show to be involved in
the TIMP-2 interaction. Thus, in the absence of the three-dimensional
structure of TIMP-2 bound to the gelatinase A C domain, this
mutagenesis approach provides convincing but nonetheless indirect
evidence that these lysine and arginine residues form important
elements of the interaction surface shown in Fig.
5.
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Our data are consistent with those of Friedman et al. (25), who found that a C domain deletion mutant that terminates at Gly454, 15 residues down from Cys440 at the start of the domain (see Fig. 2), lost TIMP-2 binding. A chimera of module I that replaced up to Pro474 with the corresponding region from stromelysin-1 was recently found by Butler et al. (22) to retain TIMP-2 binding. Another chimera that replaced all of module IV and part of module III from Lys568 to Cys631 lost TIMP-2 binding (22). From these data, the authors concluded that the TIMP-2 binding site was located between Lys568 and the carboxyl-terminal Cys631. However, a common problem of chimera studies is structural perturbations at the chimera interfaces, which may also have accounted for the loss of TIMP-2 binding. Indeed, we have found TIMP-2 binding residues outside the region implicated by the chimera studies. Thus, our data considerably refine these previous analyses by identifying the role played by individual basic residues in both modules III and IV and reveal an important contribution to TIMP-2 binding by residues amino-terminal to the Lys566-Lys-Lys568 triad.
Unlike domain chimeras, in which entire -sheet packing faces are
replaced, alanine is considered an innocuous substitution that
generally does not produce steric clashes (42). Indeed, one triple
alanine mutation (K566A/K568A/K575A) showed TIMP-2 binding properties
that were essentially identical to those of the wild-type C domain.
Thus, the introduction of three mutations does not by itself globally
destabilize the domain. Although structural causes cannot be
definitively excluded as an explanation for the reduced TIMP-2 affinity
observed at some mutation sites, the mutant alanines would not support
an ionic interaction potential with TIMP-2 partner residues. If, as our
data suggest, certain basic residues of the C domain are involved in
salt bridge formation with anionic TIMP-2 residues, the removal of the
cationic sidechain would leave an unpaired anion on the TIMP-2 surface
during binding. This would impose a penalty in free energy for burial
into the contact face of an acidic unpaired charge or an associated
water molecule. Clearly, the nature of the salt bridges characterizing the TIMP-2 binding interaction is complex with cooperative and differential roles played by the individual basic residues. Thus, simply reducing the net positive charge of a cluster was not sufficient to weaken the TIMP-2 interaction. Rather, the role of individual basic
residues in TIMP-2 recognition is position-sensitive. This is likely
related to structural considerations and distance constraints imparted
by the anionic carboxyl-terminal TIMP-2 tail. Potentially, some of the
less important TIMP-2-binding basic residues may play more important
roles in binding TIMP-4, which competes with TIMP-2 for gelatinase A C
domain binding and possibly activation (14). This hypothesis is now
under investigation in our laboratory.
Almost certainly, other surrounding nonbasic residues are required for TIMP-2 binding that will also contribute to the free energy of association. Indeed 1.0 M NaCl does not dissociate TIMP-2 from C domain-affinity columns, nor does it prevent TIMP-2 interaction from occurring in the plate assays (not shown). Thus, due to the large surface area of the TIMP-2 binding site, a pronounced effect on TIMP-2 binding by single mutations is predicted to be relatively small. Nonetheless, the observations that single point mutations at positions 547 and 617 produced such impressive shifts in the apparent Kd values of the TIMP-2 interaction and that the two double alanine mutations of Lys610/Lys617 and Lys558/Arg561 alone rendered any TIMP-2 binding undetectable, together reveal the importance of salt bridges involving some of these residues in the TIMP-2 interaction. Additional mutagenesis is needed to further characterize the binding interaction and to unequivocally assign binding roles for all contact residues. However, the combinations of mutants utilized in this investigation have proven effective in identifying an important role for salt bridge formation in TIMP-2 docking to the gelatinase A hemopexin C domain and for refining the location of the TIMP-2 binding site.
A position-sensitive interaction with TIMP-2 was demonstrated for the
cationic residues Lys547, Lys549,
Lys550, Lys558, Arg561, and
Lys575 on module III. Residues in this cluster also form a
potential heparin-binding site (34) and form the three
BX7B motifs in module III. From characterization
of the single alanine mutants at positions Lys547,
Lys549, and Lys550, the lack of effect on
TIMP-2 binding by K549A indicates that this residue lies at the border
or just outside the TIMP-2 binding surface (see Fig. 5). The more
marked alteration in TIMP-2 binding by K547A alone and by K550A in the
triple mutant protein K550A/K566A/K568A indicates that
Lys547 forms the dominant TIMP-2 recognition residue in
this cluster, with Lys550 playing a minor role. Also in
this cluster is Arg561, which when mutated to alanine
together with Lys558, displays strongly reduced TIMP-2
binding. In contrast, the nearby Lys575 does not appear to
play a role in the TIMP-2 interaction. Three-dimensional structural
analysis provides a possible explanation for these effects and suggests
an important role in TIMP-2 binding for Arg561 rather than
Lys558. Lys558 lies on the underside of the
domain, and the -strand commencing here makes a turn near
Lys566-Lys-Lys568 on the upper domain surface,
where Lys566 forms a BX7B motif with
Lys558 [Lys558-Lys566]. The
antiparallel
-strand then returns to Lys575 on the
underside of the domain and at the end of another consecutive BX7B repeat
[Lys567-Lys575]. The tertiary fold packs
Lys575 near Lys558 on the lower domain surface.
Although Arg561 is 14 residues distant from
Lys575 in the primary structure, in the tertiary structure,
these residues lie close to each other. As the lower panel
in Fig. 5 shows, Arg561 is on the outer rim of the upper
surface of the domain. Lys575, which is not implicated in
TIMP-2 binding, is interposed between it and Lys558 on the
lower surface. Thus, from analysis of the binding data for the
K558A/R561A mutant protein in the context of this structural information, we consider that the primary effect on TIMP-2 binding by
this double mutation is through substitution of the Arg561.
Although not ruled out by these mutations, a contribution to TIMP-2
binding by Lys558 is a possibility that is effectively
discounted by these structural considerations.
The function of the Lys566-Lys-Lys568 triad in
TIMP-2 binding is enigmatic. These sites lie on the upper surface of
the domain close to a turn connecting two antiparallel -strands. The
influence of this cationic triad on TIMP-2 binding was first studied by a double mutation (K566A/K568A), designed to reduce the overall charge
of this cluster. We then added mutations of BX7B
motifs onto the K566A/K568A background. Replacement of two of the
lysine residues in Lys566-Lys-Lys568 with
alanine did not disrupt TIMP-2 binding. Although substitution of
Lys567 was not performed, an antipeptide antibody to this
sequence also did not block the TIMP-2 interaction. Thus, our studies
do not support the recent report that
Lys566-Lys-Lys568 is important in the TIMP-2
interaction (22). However, when the K566A/K568A mutations were combined
with other substitutions (K550A and K617A), a marked reduction in
TIMP-2 interaction resulted. This reveals a small contribution to
TIMP-2 binding by Lys566-Lys-Lys568 that is not
strong enough to be manifest without other changes to the contact
surface. From its topographical location in relation to
Lys550, which also appears to only make a weak interaction
with TIMP-2, this outlines a small zone of weak TIMP-2 contact that may
account for the minor direct effects on TIMP-2 binding by mutations of these residues alone. As Fig. 5 shows, this weak binding zone (shown by
the hatched pattern) is at one end of the main TIMP-2 binding surface that is defined by the more effective mutation sites
(shown within the dotted perimeter).
Although heparin has been shown to bind the C domain (33, 34) and to potentiate progelatinase A activation on the cell surface (22, 43), the heparin binding residues and their relationship to TIMP-2 binding have yet to be determined. Understanding the nature of the simultaneous heparin and TIMP-2 binding interactions is likely to be complex. Further use of the mutants generated here will be invaluable for our ongoing studies to understand these cooperative interactions. Heparin interaction may involve the potential heparin binding sites Lys547-Asn-Lys-Lys550 and 566Lys-Lys-Lys568 (34). Notably, our data reveal that two residues in the first motif (Lys547 and Lys550) appear to play a role in the TIMP-2 interaction and so may no longer interact with heparin after TIMP-2 has bound to the domain. This possibility supports the potential involvement in heparin binding of Lys566-Lys-Lys568 to increase the rate of gelatinase A activation. The Lys547-Asn-Lys-Lys550 sequence also contributes to the first BX7B motif, and Lys566-Lys-Lys568 contributes to both the second and third BX7B motifs. In the cell adhesion molecule RHAMM (receptor for HA-mediated motility), the BX7B motif has been shown to bind hyaluronan (32), but recent evidence has cast doubt on this function (44). Indeed, we have found no evidence for hyaluronan binding by the C domain,3 but this may be due to the presence of an acidic residue in each of these repeats that reduces hyaluronan binding. Because the BX7B motifs also do not appear to play a critical role in TIMP-2 binding, the significance of these motifs in the gelatinase A C domain remains unclear.
The marked clustering of positive charges to one face of the gelatinase A C domain introduces a considerable dipole moment to the molecule. Thus, a related role for these cationic residues may be to correctly orientate the TIMP-2 contact face of the C domain with the anionic surface of TIMP-2 immediately prior to protein contact. In the active enzyme, these ionic interactions may thereby favor TIMP-2 binding first to the C domain rather than to the active site potentially explaining how TIMP-2 can bind the C domain despite the stronger binding interaction with the active site (45). Indeed, the recently published three-dimensional structure of full-length TIMP-2 bound to the active site of MT1-MMP (46) shows that the TIMP-2 carboxyl-terminal tail exhibits considerable disorder. Such molecular flexibility of the anionic tail may allow initial contact and molecular alignment with the gelatinase A C domain as a first step prior to full protein contact and stabilization of the bound complex. In this regard, we observed an approximate 0.5-fold reduction in the relative amount of TIMP-2 bound with the K617A and K547A mutant C domains in addition to a reduction in apparent Kd values. In a two-part binding reaction, this may indicate that one of the two bound states of TIMP-2 on the C domain is perturbed by these mutations. Indeed, biphasic kinetics for binding of TIMP-2 to progelatinase A have been reported (46).
Locating on the three-dimensional structure of the gelatinase A C
domain the mutation sites that disrupted TIMP-2 binding, compared with
those that did not, identifies, with higher precision than previous
deletion and chimera studies, the general position of the TIMP-2
contact interface. Thus, TIMP-2 binds the upper surface of the
gelatinase A C domain, at the junction of hemopexin modules III and IV,
to form an elongated binding surface on the outside rim of the domain
(Fig. 5). This region also includes a zone of weaker TIMP-2 interaction
between Lys550 and Lys566/Lys568
(Fig. 5, hatched pattern). Notably, this is unlike many
-propeller structures, which bind ligands at the top of the
-propeller near the pseudosymmetry axis (47, 48). The asymmetrical
localization of the TIMP-2 binding site away from the central axis of
the C domain has several implications for the function of gelatinase A
and the bound TIMP-2, particularly in terms of cellular binding and
activation by MT-MMPs. By analogy with the full-length porcine collagenase-1 structure (36), our study indicates that the bound TIMP-2
would lie away from the active site, on the opposite side of the
molecule from the fibronectin type II-like repeats that form the
collagen binding domain of gelatinase A (40). This orientation would
likely preclude the inhibition in cis of the gelatinase A
active site by the C domain-bound TIMP-2. At the same time, it would
permit interaction of the NH2-inhibitory domain of the
bound TIMP-2 with the active site of MT-MMPs that can act as cell
receptors for gelatinase A (20-23). Free access by a second MT-MMP
that is proposed to initially cleave the gelatinase A prodomain (24,
43) at Asn37-Leu38 would also not be impeded by
this spatial arrangement. Inhibition of gelatinase A activity by TIMP-2
would therefore require a second TIMP molecule that can be
simultaneously bound by the active site of gelatinase A (12, 13),
rather than through inhibition by the C domain-bound TIMP-2 as some
authors suggest. This complex ternary interaction also requires
stabilization of the active site-bound TIMP-2 with the C domain of
gelatinase A (46) at a second site. Indeed, we have recently found that
the NH2-domain of TIMP-2 binds with low affinity to the
gelatinase A C domain (24, 27).3 In this regard, a
gelatinase A C domain chimera generated by Butler et al.
(22) that replaced most of hemopexin module I up to Pro474
with the corresponding region of stromelysin-1 still retained TIMP-2
binding properties. In consideration of our studies, the stabilization
site for the gelatinase A inhibitory TIMP-2 molecule might therefore
lie on the rim of hemopexin module II of the gelatinase A C domain.
Lastly, these studies are consistent with the notion that the
carboxyl-terminal tail of TIMP-2 binds to the gelatinase A C domain.
Whether TIMP-2 binding to the gelatinase A C domain masks the growth
factor effects of the TIMP-2 carboxyl domain is an intriguing prospect
that the mutant domains generated here should prove useful in helping
to address.
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FOOTNOTES |
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* This work was supported by Grant 006388 from the National Cancer Institute of Canada.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.
§ Recipient of a Medical Research Council of Canada Clinician Scientist Award. To whom correspondence should be addressed: University of British Columbia, 2199 Wesbrook Mall, Vancouver, British Columbia V6T 1Z3, Canada. Tel.: 604-822-2958; Fax: 604-822-3561; E-mail: overall{at}interchange.ubc.ca.
The abbreviations used are: MMP, matrix metalloproteinase; C domain, carboxyl-terminal domain; MT, membrane-type; PBS, phosphate-buffered saline; TIMP, tissue inhibitor of metalloproteinases.
2 C. M. Overall, H. Tschesche, and A. King, unpublished observations.
3 C. M. Overall, U. M. Wallon, G. A. McQuibban, E. Tam, H. F. Bigg, C. J. Morrison, Y. DeClerck, and A. E. King, manuscript in preparation.
4 C. R. Roberts and C. M. Overall, unpublished data.
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