(Received for publication, September 17, 1996, and in revised form, November 20, 1996)
From the Department of Biochemistry, University of Louisville School of Medicine, Louisville, Kentucky 40292
Dissociation of Ca2+ from human interstitial collagenase induced either by chelation with EGTA or by dilution resulted in loss of enzyme activity, a red shifted emission maximum from 334 to 340 nm and quenching of protein fluorescence by 10% at 340 nm. Circular dichroism indicated that secondary structure was unaffected by EGTA. Ca2+ binding to the EGTA-treated enzyme as assessed by fluorescence was cooperative (Hill coefficient, 2.9; 50% saturation at 0.4 mM Ca2+). The dependence of catalytic activity on [Ca2+] was also cooperative (Hill coefficient, 1.7-2.0; midpoint [Ca2+], 0.2 mM). The Ca2+-reconstituted protein was indistinguishable from the untreated enzyme by activity and fluorescence measurements. These results demonstrate that removal of Ca2+ from full-length collagenase generates a catalytically incompetent, partially unfolded state with native secondary structure but altered tertiary structure characterized by exposure of at least one tryptophyl residue to a more polar environment.
The matrix metalloproteinases mediate remodeling of extracellular matrix in healthy and diseased tissue (1, 2). Sequence analysis of the MMPs,1 also referred to as the metzincin proteinase family (3), reveals a characteristic domain structure. The smallest MMP, matrilysin, consists of 19-kDa catalytic domain with an active site zinc ion coordinated within a conserved HEXXHXXGXXH motif. Proteolytic latency is maintained by an N-terminal propeptide of approximately 10 kDa in which a conserved cysteine is coordinated to the catalytic zinc (4). The larger members of the family, including interstitial collagenases, stromelysins, and gelatinases, possess additional functional and structural domains. In particular, collagenases have a C-terminal domain of about 30 kDa that exhibits sequence homology with hemopexin and vitronectin (5). This so-called pexin domain is essential for the expression of collagenolytic activity; without it, the enzyme retains general peptidase activity but does not hydrolyze native collagen (6). Matrilysin and mutant forms of collagenase and stromelysin possessing only a catalytic domain contain a second zinc ion (7-9). This structural zinc is absent from the full-length forms of stromelysin-1 and gelatinase A (10).
Calcium ions are also an essential components of the MMPs (11, 12). Crystallographic analysis of the catalytic domain reveals three bound Ca2+ in fibroblast collagenase (13, 14) and two in the neutrophil enzyme (15). Crystallography of full-length porcine MMP-1 also shows three Ca2+ in the catalytic portion and two Ca2+ in the pexin domain (16). Ca2+ undoubtedly binds to similar sites in the pexin domain of human collagenase because the binding sites are conserved. Previous studies suggest that Ca2+ stabilizes an active conformation of the MMP that is more stable to denaturants (17) and less susceptible to proteolysis (18). However, the precise stereochemical basis for Ca2+ stabilization is unknown because a three-dimensional model of a Ca2+-free MMP is not available for comparison.
To provide additional insight into the relationship between MMP structure and calcium binding, we correlated the effect of Ca2+ on catalytic activity and protein structure using intrinsic protein fluorescence and circular dichroism as structural indicators. Ca2+ concentration was manipulated either by the chelating agent EGTA or by dilution. EGTA was chosen because its affinity for Ca2+ is comparable with that of EDTA,2 although its affinity for Zn2+ is much lower than that of EDTA (19). The following data document that Ca2+ binding to collagenase is cooperative and associated with changes in tertiary but not secondary structure. Furthermore, these changes were independent of the method used to alter calcium concentration.
A recombinant human fibroblast collagenase expression vector was generously supplied by Dr. G. McGeehan, Glaxo-Wellcome (Research Triangle Park, NC). The recombinant protein, which lacks the propeptide, was expressed in Escherichia coli and purified as described (20). Native procollagenase was purified from culture medium of human umbilical vein endothelial cells (20). The proenzyme was activated with trypsin (20 ng of trypsin/1 µg of proMMP-1) for 1 h at 37 °C (21); trypsin was subsequently inactivated with soybean trypsin inhibitor. The substrate peptide Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 (DnpS) was synthesized by Stack and Gray (22); Ac-Pro-Leu-Gly-(2-mercapto-4-methyl-pentanoyl)-Leu-Gly-OEt (TPS) was purchased from Bachem (King of Prussia, PA). Type I collagen from calf skin, soybean trypsin inhibitor, 4,4-dithiopyridine, MOPS, and EGTA were from Sigma. CaCl2 2H2O was from EM Science (Cherry Hill, NJ); it contained 6 ng zinc/g. Brij 35 (prepared and packaged under nitrogen) was from Pierce, and trypsin (sequencing grade) was from Promega (Madison, WI). Zinc reference standard solution was from Fisher.
MethodsMMP activity was measured with DnpS, TPS, or
collagen as substrate. Hydrolysis of DnpS by rMMP-1 was assessed by
high pressure liquid chromatography as described (22). Hydrolysis of
TPS by HUVEC MMP-1 was measured in a continuous spectrophotometric
assay using 4,4-dithiopyridine to trap the thiol peptide product,
HSCH(iBu)-Leu-Gly-OEt (23). Assays were conducted in a 96-well
UV-transparent microtiter plate with a Spectramax 250 UV-visible plate
reader (Molecular Devices Corp., Sunnyvale, CA) set at 324 nm to record
the kinetics of substrate hydrolysis. Collagenolytic activity was
estimated under the conditions of Terato et al. (24).
Collagen degradation products were separated by polyacrylamide gel
electrophoresis (25); the fraction of degraded
-chains was estimated
by densitometric analysis of the Coomassie Blue-stained gels (26, 27).
Fluorescence spectra were recorded with an SLM-Aminco SPF-500C
spectrofluorometer interfaced to an IBM personal computer. All spectra
have buffer blanks subtracted, and fluorescence intensities are
presented in arbitrary units. Difference spectra were generated by
subtracting the relevant absolute spectra using software supplied by
SLM-Aminco. CD spectra were recorded with a Jasco J-710
spectropolarimeter as described previously (20). All assays and spectra
were obtained at 25 °C. Zinc analyses were conducted by furnace
atomic absorption analysis.
To assess the effect of Ca2+ binding on rMMP-1
secondary structure, we recorded the low UV CD spectrum of proMMP-1 in
the presence and the absence of the chelating agent, EGTA. As shown in
Fig. 1A, EGTA had no effect on the CD
spectrum of the enzyme. The addition of Ca2+ to the
EGTA-treated protein also elicited no observable change in CD spectrum.
Similar results were obtained with the recombinant MMP-1 (data not
shown). Thus, we conclude Ca2+ binding to MMP-1 resulted in
no global changes in secondary structure.
We also measured the emission spectrum of rMMP-1 in the presence of 5 mM CaCl2 before and after the addition of 6 mM EGTA to assess Ca2+-dependent changes in tertiary structure. Fig. 1B shows that the addition of EGTA resulted in decreased fluorescence intensity of 10% at 340 nm and a red shift in the emission maximum from 334 to 340 nm (compare spectra 1 and 2). Similar changes in emission intensity were observed when HUVEC procollagenase was diluted into Ca2+-free buffer (Fig. 1B, spectrum 4) and then supplemented with added Ca2+ (Fig. 1B, spectrum 3). This suggests that the fluorescence changes in spectra 1 and 2 result from dissociation of Ca2+ induced by the chelating agent. Lack of an obvious shift in emission maximum in comparing spectra 3 and 4 may result from the presence of the propeptide. This portion of the protein contains one tryptophan, which could have obscured the shift in emission maximum associated with Ca2+ dissociation from the enzyme lacking the propeptide.
The emission characteristics of the quenched residue (shown in Fig. 1B, spectrum 5) were assessed by subtracting the spectrum measured in the presence of EGTA (spectrum 2) from that obtained in its absence (spectrum 1). The maximum in the difference spectrum 5 at 320 nm indicates that the quenched fluorophores are predominantly tryptophyl residues that reside in a hydrophobic environment (28).
The contribution of tyrosyl residues to rMMP-1 fluorescence was
ascertained by subtracting the emission spectrum obtained with
excitation at 297 nm (where Trp alone absorbs) from the spectrum obtained with excitation at 280 nm (Trp and Tyr absorb). The resulting difference spectra (Fig. 2) allow comparison of the
emission properties of tyrosyl residues in the Ca2+-free
and Ca2+-bound forms of the enzyme. Each spectrum exhibits
a maximum near 315 nm, which is strongly red shifted from that expected
for tyrosine in aqueous solution (28). A small decrease in emission
intensity was observed, suggesting that the
Ca2+-dependent conformational differences are
sensed by tyrosyl as well as tryptophyl residues.
Titration data illustrating the dependence of fluorescence and
catalytic activity on [Ca2+] are shown in the Hill plots
of Fig. 3. Ca2+ binding as assessed by
changes in fluorescence was cooperative (Hill coefficient, 2.9; 50%
saturation at 0.43 mM Ca2+). When the
Ca2+ dependence of rMMP-1 activity was assayed with a
peptide substrate, the resulting Hill plot also indicated cooperative
Ca2+ binding (Hill coefficient, 1.7; 50% saturation at
0.23 mM [Ca2+]). When the
[Ca2+] was manipulated by dilution rather than EGTA,
cooperative Ca2+ binding to native HUVEC MMP-1 was also
observed (Hill coefficient, 2.0; 50% saturation at 0.20 mM
Ca2+). Because similar results were obtained whether
[Ca2+] was altered by EGTA or dilution, we conclude that
the effects of the chelating agent result from Ca2+
dissociation from the enzyme rather than removal of catalytic Zn2+.
To determine if the effects of Ca2+ on activity and fluorescence were reversible, we compared the catalytic activity of the recombinant enzyme with DnpS and collagen prior to Ca2+ removal, after Ca2+ removal with EGTA, and after reconstitution with Ca2+. Previous reports (11, 12) indicate that prolonged incubation of collagenase with EDTA resulted in removal of zinc and irreversible denaturation of the protein. The data in Table I show that activity against both DnpS and collagen was completely lost on treatment with EGTA. Loss of activity and fluorescence changes were reversed by adding Ca2+, even 24 h after removal of the metal ion.
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The present studies, conducted with full-length rMMP-1 that includes both the catalytic and pexin domains, demonstrate that acquisition of catalytic activity by collagenase is coupled to protein conformational changes induced by Ca2+ binding. Binding was cooperative as assessed by both structural and activity changes. The observation that a lower Ca2+ concentration is required for catalytic activity compared with development of the fluorescence changes suggests that fewer binding sites influence catalysis, whereas the fluorescence changes involve sites other than those necessary for catalytic efficiency. Our studies with the full-length protein extend previous studies conducted with the catalytic domain alone. Lowry et al. (17) showed that both Ca2+ and Zn2+ stabilized a recombinant collagenase catalytic domain against denaturation by GdnHCl, and Housley et al. (18) reported that Ca2+ stabilized the catalytic domain of recombinant stromelysin against heat denaturation. In addition, activation of prostromelysin-1 by an organomercurial in the presence of 0.1 mM Ca2+ resulted in autolysis that could be prevented with 5 mM Ca2+ (18). These data indicate that Ca2+ stabilizes a compact protein structure that is less susceptible to denaturation and proteolysis. Our results show that Ca2+ binding to full-length rMMP-1 results in local conformational change(s) that do not detectably alter the secondary structure of the protein but do change tertiary structure as reflected in the environment of tryptophyl and tyrosyl residues. The change in tertiary structure in the absence of secondary structural changes suggests that Ca2+ removal may produce a "molten globule-like" structure (20).
The emission maximum of denatured MMP-1 is at 356 nm (20), characteristic of tryptophan in water, whereas the emission maximum of the Ca2+-deficient enzyme shifts only to 340 nm. Thus the change in tryptophyl environment brought about by Ca2+ release is less drastic than complete unfolding. In our study of GdnHCl-induced denaturation of rMMP-1, we observed a folding intermediate with an emission maximum at 340 nm at 1 M denaturant (20). However, it is unlikely that the Ca2+-free form of MMP-1 in the present study is the same as this intermediate because in 1 M GdnHCl, there was an increase, rather than a decrease, in emission intensity.
MMP-1 contains one tryptophyl residue in the propeptide, three in the
catalytic domain and four in the pexin domain. Assignment of the
fluorescence changes to particular residues is of course impossible
from the data at hand. Lovejoy et al. (13) suggested that in
19-kDa collagenase one of the calcium ions and the structural zinc may
stabilize a surface loop by fastening it to two strands. Neither
the loop nor the two strands contain tyrosyl or tryptophyl side chains
that might be directly affected by Ca2+. Thus, it is likely
that any conformational change induced by Ca2+ binding at
this site is propagated to other regions of the structure. The two
Ca2+ in the pexin domain of pig collagenase also appear to
link structural domains together:
-sheet-1 to sheet 3 and
-sheet-4 to sheet 2 (16). Two of the four pexin tryptophyl residues
are located within these sheets; Trp349 is in sheet 2, and
Trp398 is in sheet 3. Thus, it is reasonable to assign at
least a portion of the Ca2+-dependent
fluorescence change to them. In addition, both tryptophyls are close in
the primary structure to tyrosyl residues [YW349A and
YW398RY (29)]. Variation in the relationship between these
groups should influence the efficiency of excitation energy transferred from tyrosyl to tryptophyl residues. However, the quenching of tyrosyl
emission observed on Ca2+ dissociation was accompanied by
decreased, rather than increased, tryptophyl emission, as would be
expected if altered transfer efficiency were the sole basis for the
altered fluorescence.
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