Inhibition of the Tumor Necrosis Factor-α-converting Enzyme by Its Pro Domain
2004; Elsevier BV; Volume: 279; Issue: 30 Linguagem: Inglês
10.1074/jbc.m401311200
ISSN1083-351X
AutoresPatricia Gonzales Gil, Ariel Solomon, Ann B. Miller, M. Anthony Leesnitzer, Irit Sagi, Marcos E. Milla,
Tópico(s)Peptidase Inhibition and Analysis
ResumoTumor necrosis factor-α-converting enzyme (TACE) is a disintegrin metalloproteinase that processes tumor necrosis factor and a host of other ectodomains. TACE is biosynthesized as a zymogen, and activation requires the removal of an inhibitory pro domain. Little is known about how the pro domain exerts inhibition for this class of enzymes. To study the inhibitory properties of the pro domain of TACE, we have expressed it in isolation from the rest of the protease. Here we show that the TACE pro domain (TACE Pro) is a stably folded protein that is able to inhibit this enzyme. TACE Pro inhibited the catalytic domain of TACE with an IC50 of 70 nm. In contrast, this inhibitory potency decreased over 30-fold against a TACE form containing the catalytic plus disintegrin/cysteine-rich domains (IC50 greater that 2 μm). The disintegrin/cysteine-rich region in isolation also decreases the interaction of TACE Pro with the catalytic domain. Surprisingly, we found that the cysteine switch motif located in TACE Pro was not essential for inhibition of the enzymatic activity of TACE; the pro domain variant C184A showed the same inhibitory potency against both TACE forms as wild type TACE Pro. X-ray absorption spectroscopy experiments indicate that binding of TACE Pro to the catalytic domain does include ligation of the catalytic zinc ion via the sulfur atom of its conserved Cys184 residue. Moreover, the binding of TACE Pro to the catalytic zinc ion partially oxidizes the catalytic zinc ion of the enzyme. Despite this, the nature of the interaction between the pro and catalytic domains of TACE is not consistent with a simple competitive model of inhibition based on cysteine switch ligation of the zinc ion within the active site of TACE. Tumor necrosis factor-α-converting enzyme (TACE) is a disintegrin metalloproteinase that processes tumor necrosis factor and a host of other ectodomains. TACE is biosynthesized as a zymogen, and activation requires the removal of an inhibitory pro domain. Little is known about how the pro domain exerts inhibition for this class of enzymes. To study the inhibitory properties of the pro domain of TACE, we have expressed it in isolation from the rest of the protease. Here we show that the TACE pro domain (TACE Pro) is a stably folded protein that is able to inhibit this enzyme. TACE Pro inhibited the catalytic domain of TACE with an IC50 of 70 nm. In contrast, this inhibitory potency decreased over 30-fold against a TACE form containing the catalytic plus disintegrin/cysteine-rich domains (IC50 greater that 2 μm). The disintegrin/cysteine-rich region in isolation also decreases the interaction of TACE Pro with the catalytic domain. Surprisingly, we found that the cysteine switch motif located in TACE Pro was not essential for inhibition of the enzymatic activity of TACE; the pro domain variant C184A showed the same inhibitory potency against both TACE forms as wild type TACE Pro. X-ray absorption spectroscopy experiments indicate that binding of TACE Pro to the catalytic domain does include ligation of the catalytic zinc ion via the sulfur atom of its conserved Cys184 residue. Moreover, the binding of TACE Pro to the catalytic zinc ion partially oxidizes the catalytic zinc ion of the enzyme. Despite this, the nature of the interaction between the pro and catalytic domains of TACE is not consistent with a simple competitive model of inhibition based on cysteine switch ligation of the zinc ion within the active site of TACE. The tumor necrosis factor-α-converting enzyme (TACE 1The abbreviations used are: TACE, tumor necrosis factor-α-converting enzyme; TACE Pro, TACE pro domain; Gdn-HCl, guanidinium hydrochloride; XAS, X-ray absorption spectroscopy; XANES, x-ray absorption near edge spectra; EXAFS, extended x-ray absorption fine structure; MMP, matrix metalloproteinase. or ADAM 17) is a zinc metalloproteinase that cleaves the precursor, membrane-bound form of tumor necrosis factor-α and other ectodomains (1Moss M.L. Jin S.-L.C. Milla M.E. Burkhart W. Carter H.L. Chen W.-J. Clay W.C. Didsbury J.R. Hassler D. Hoffman C.R. Kost T.A. Lambert M.H. Leesnitzer M.A. McCauley P. McGeehan G. Mitchell J. Moyer M. Pahel G. Rocque W. Overton L.K. Schoenen F. Seaton T. Su J.-L. Warner J. Willard D. Becherer J.D. Nature. 1997; 385: 733-736Google Scholar, 2Black R.A. Rauch C.T. Kozlosky C.J. Peschon J.J. Slack J.L. Wolfson M.F. Castner B.J. Stocking K.L. Reddy P. Srinivasan S. Nelson N. Boiani N. Schooley K.A. Gerhart M. Davis R. Fitzner J.N. Johnson R.S. Paxton R.J. March C.J. Cerretti D.P. Nature. 1997; 385: 729-733Google Scholar, 3Peschon J.J. Slack J.L. Reddy P. Stocking K.L. Sunnaborg S.W. Lee D.C. Russell W.E. Castner B.J. Johnson R.S. Fitzner J.N. Boyce R.W. Nelson N. Kozlosky C.J. Wolfson M.F. Rauch C.T. Cerretti D.P. Paxton R.J. March C.J. Black R.A. Science. 1998; 282: 1279-1280Google Scholar). TACE is a member of a family of proteases known as the ADAMs (adisintegrin and metalloproteinase). ADAMs are multidomain proteins typically comprising a pro domain, metalloproteinase domain, a disintegrin/cysteine-rich domain, transmembrane domain, and a cytoplasmic tail (4Wolfsberg T.G. Primakoff P. Myles D.G. White J.M. J. Cell Biol. 1995; 131: 275-278Google Scholar, 5Schlondorff J. Blobel C.P. J. Cell Sci. 1999; 112: 3603-3617Google Scholar). ADAMs are synthesized as latent precursors and later converted to the mature enzyme, lacking the pro domain, by furin or a furin-like enzyme in the secretory pathway (6Endres K. Anders A. Kojro E. Gilbert S. Fahrenholz F. Postina R. Eur. J. Biochem. 2003; 270: 2386-2393Google Scholar, 7Kang T. Zhao Y.G. Pei D. Sucic J.F. Sang Q.X.A. J. Biol. Chem. 2002; 277: 25583-25591Google Scholar). It has been shown previously that the pro domain of TACE seems to act as an inhibitor of this enzyme, because the activity of TACE was only recovered upon its removal (8Milla M.E. Leesnitzer M.A. Moss M.L. Clay W.C. Carter H.L. Miller A.B. Su J.-L. Lambert M.H. Willard D.H. Sheeley D.M. Kost T.A. Burkhart W. Moyer M. Blackburn R.K. Pahel G.L. Mitchell J.L. Hoffman C.R. Becherer J.D. J. Biol. Chem. 1999; 274: 30563-30570Google Scholar). TACE Pro includes a cysteine switch box (PKVCGY186), a feature present in most metzincins, including matrix metalloproteinases and ADAMs. It has been proposed that the pro domains of metzincins act as inhibitors of their catalytic domains through a mechanism that involves ligation of the cysteinyl thiol within the cysteine switch box to the zinc ion in the active site (9van Wart H. Birkedal-Hansen B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5578-5581Google Scholar, 10Galazka G. Windsor L.J. Birkedal-Hansen H. Engler J.A. Biochemistry. 1996; 35: 11221-11227Google Scholar, 11Galazka G. Windsor L.J. Birkedal-Hansen H. Engler J.A. Biochemistry. 1999; 38: 1316-1322Google Scholar). The cysteine switch present in TACE Pro did appear to be important for inhibition of this enzyme, because thiol-modifying reagents such as 4-aminophenylmercuric acetate and octylthioglucoside promoted pro domain release from the catalytic domain and, therefore, enzyme activation (8Milla M.E. Leesnitzer M.A. Moss M.L. Clay W.C. Carter H.L. Miller A.B. Su J.-L. Lambert M.H. Willard D.H. Sheeley D.M. Kost T.A. Burkhart W. Moyer M. Blackburn R.K. Pahel G.L. Mitchell J.L. Hoffman C.R. Becherer J.D. J. Biol. Chem. 1999; 274: 30563-30570Google Scholar). TACE Pro also serves a second function: it is essential for the secretion of functional enzyme. In insect cells, a recombinant form of this enzyme lacking the pro domain failed to be secreted and was extensively degraded intracellularly (8Milla M.E. Leesnitzer M.A. Moss M.L. Clay W.C. Carter H.L. Miller A.B. Su J.-L. Lambert M.H. Willard D.H. Sheeley D.M. Kost T.A. Burkhart W. Moyer M. Blackburn R.K. Pahel G.L. Mitchell J.L. Hoffman C.R. Becherer J.D. J. Biol. Chem. 1999; 274: 30563-30570Google Scholar). Similar results have been reported for other members of the ADAM family (12Anders A. Gilbert S. Garten W. Postina R. Fahrenholz F. FASEB J. 2001; 15: 1837-1839Google Scholar, 13Loechel F. Overgaard M.T. Oxvig C. Albrechtsen R. Wewer U.M. J. Biol. Chem. 1999; 274: 13427-13433Google Scholar). At first approximation, this is similar to the secreted bacterial serine proteases subtilisin and α-lytic protease, in which the pro domain accelerates the folding of the catalytic domain by several orders of magnitude, apparently by lowering the conformational energy barrier between the unfolded and native states (14Baker D. Sohl J.L. Agard D.A. Nature. 1992; 356: 263-265Google Scholar, 15Cunningham E.L. Jaswal S.S. Sohl J.L. Agard D.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11008-11014Google Scholar, 16Strausberg S. Alexander P. Wang L. Schwarz F. Bryan P. Biochemistry. 1993; 32: 8112-8119Google Scholar, 17Eder J. Rheinnecker M. Fersht A.R. Biochemistry. 1993; 32: 18-26Google Scholar). Here we show that the isolated pro domain of TACE is a potent inhibitor of the catalytic domain of this enzyme. TACE Pro is a less effective inhibitor of a form of TACE comprising the catalytic plus disintegrin/cysteine-rich domains, suggesting a role for the disintegrin/cysteine-rich domain in pro domain removal and enzyme activation. We also show that the cysteine switch of TACE, although present in the zymogen form, is not required for the interaction between the pro and catalytic domains of TACE. Plasmid Constructs—A plasmid for expression of the pro domain of TACE in Escherichia coli, lacking the signal peptide, was constructed using the vector pRSET B (Invitrogen). A DNA fragment encoding residues Asp22 to Arg214 with an NdeI site at the 5′-end and a BamHI site at the 3′-end was generated by polymerase chain reaction amplification using a previously reported pFastBac1 TACE plasmid as a template (8Milla M.E. Leesnitzer M.A. Moss M.L. Clay W.C. Carter H.L. Miller A.B. Su J.-L. Lambert M.H. Willard D.H. Sheeley D.M. Kost T.A. Burkhart W. Moyer M. Blackburn R.K. Pahel G.L. Mitchell J.L. Hoffman C.R. Becherer J.D. J. Biol. Chem. 1999; 274: 30563-30570Google Scholar). The fragment was inserted at the BamHI and NdeI sites of pRSET B to obtain TACE Pro. TACE Pro C184A was made by cassette mutagenesis. The cassette was designed with EcoRV- and AccI-compatible ends for insertion into TACE Pro and carried a Cys to Ala mutation at position 184. Additional constructs for expression of double alanine mutants in the TACE Pro cysteine switch region (PKVCGYLKDVD190) were made by the same cassette mutagenesis approach. They carried a Cys to Ala mutation in position 184 and an additional alanine substitution at each individual position in the cysteine switch region. Expression of Recombinant TACE Pro Proteins—E. coli BL21(DE3) electrocompetent cells were transformed with the corresponding TACE Pro plasmid and plated on LB plates containing 150 μg/ml ampicillin. After overnight incubation at 37 °C, the cells were resuspended in 1 liter of LB with 150 μg/ml ampicillin. The cells were grown at 37 °C, induced with 1 mm isopropyl-β-d-thiogalactopyranoside at an optical density of 0.6 and harvested 3 h after induction. Purification and Refolding of Wild Type and Mutant TACE Pro—The cell pellets were washed once with 20 mm Tris-HCl, pH 8, and twice with the same buffer containing 0.1% Triton X-100. Washed pellets were solubilized in 20 mm Tris-HCl, pH 8, containing 6 m Gdn-HCl and centrifuged at 26,000 × g for 30 min. The supernatant was applied to a 20-ml Ni+2-nitrilotriacetic acid column. The column was washed with 20 mm Tris-HCl, pH 8, 6 m Gdn-HCl and then with the same buffer containing 20 mm imidazole. The protein was eluted with 20 mm Tris-HCl, pH 8, 6 m Gdn-HCl, 300 mm imidazole. The eluate was dialyzed against water. Under these conditions, the protein formed a white precipitate that was then resuspended in 20 mm Tris-HCl, pH 8, 6 m Gdn-HCl to a concentration of 6 mg/ml. 50 μl of this protein solution were incubated with 950 μl of refolding buffer (FoldIt Screen Formulation 16; Hampton Research) with addition of 1 mm reduced glutathione, 0.1 mm oxidized glutathione, and 3 mm lauryl maltoside for 4 h at 4 °C with gentle stirring. After refolding, the solution was centrifuged at 10,000 × g for 10 min and filtered through a 0.22-μm filter. Finally, it was dialyzed against 250 ml of 20 mm Tris-HCl, pH 8, with 150 mm NaCl (buffer A). The final protein concentration obtained after this procedure was 4–6 μm (0.09–0.14 mg/ml). Expression and Purification of TACE Truncates R473 and R651— The construction of TACE truncates R473 (Met1–Arg473) and R651 (Met1–Arg651) and production of baculovirus in Sf9 (Spodoptera frugiperda) cells for protein production in insect cells have been described previously (8Milla M.E. Leesnitzer M.A. Moss M.L. Clay W.C. Carter H.L. Miller A.B. Su J.-L. Lambert M.H. Willard D.H. Sheeley D.M. Kost T.A. Burkhart W. Moyer M. Blackburn R.K. Pahel G.L. Mitchell J.L. Hoffman C.R. Becherer J.D. J. Biol. Chem. 1999; 274: 30563-30570Google Scholar). Logarithmically growing Trichoplusia ni cells were infected with baculovirus strains encoding R473 or R651 at a multiplicity of infection of 1. The cultures were harvested 48 h after infection, and the proteins were purified as described previously (8Milla M.E. Leesnitzer M.A. Moss M.L. Clay W.C. Carter H.L. Miller A.B. Su J.-L. Lambert M.H. Willard D.H. Sheeley D.M. Kost T.A. Burkhart W. Moyer M. Blackburn R.K. Pahel G.L. Mitchell J.L. Hoffman C.R. Becherer J.D. J. Biol. Chem. 1999; 274: 30563-30570Google Scholar). TACE Activity and Inhibition Assays—The inhibitory activity of TACE Pro was determined by an high performance liquid chromatography-based assay using the synthetic peptide dinitrophenyl-SPLAQAVRSSSR-NH2 as the substrate. TACE (R473 and R651), at a final concentration of 1 nm, was incubated with a series of TACE Pro concentrations ranging from 0 to 6 μm in buffer A. The incubation took place for 20 min at 37 °C with gentle shaking. The reaction was initiated by the addition of substrate at a final concentration of 20 μm. The reactions were incubated at 37 °C for 30 min with gentle shaking, quenched by the addition of an equal volume of 1% heptafluorobutyric acid, and filtered using polyvinyl difluoride membrane filters. The quenched reaction mixtures were applied to a 150-mm C18 column (Vydac) and resolved using a gradient of 0.1% heptaflurobutyric acid in water and 0.1% heptaflurobutyric acid in acetonitrile. The ratio of product/substrate was obtained by integration of the absorbance of the resolved peaks at 350 nm. Circular Dichroism Spectroscopy—CD spectra were recorded in an AVIV 62DS spectropolarimeter. Protein stocks were maintained at a concentration of 180 μm in buffer A containing 6 m Gdn-HCl. Those stocks were rapidly diluted 60-fold in buffer A to reach a final concentration of 3 μm of refolded protein. The spectra were taken between 208 and 260 nm using a 1-cm-path fused quartz cuvette. Scans were done in triplicate. For denaturation studies, the protein stocks were diluted into buffer A containing Gdn-HCl concentrations ranging from 0 to 7.5 m. The ellipticity at 222 nm was recorded for four independent protein dilutions at each Gdn-HCl concentration. Fluorescence Spectroscopy—The fluorescence emission scans were obtained using an AVIV ATF-105 spectrofluorometer. The samples were prepared in the same way as for CD spectroscopy. The protein samples were excited at 280 nm, and the intrinsic tryptophan emission spectra were recorded from 300 to 400 nm using a 3-mm-path cuvette. The photomultiplier tube was preset to 50% of the maximum output at a fixed excitation wavelength of 280 nm and an emission wavelength of 350 nm. For denaturation studies, the protein stocks were also diluted in buffer A containing a range of Gdn-HCl concentrations from 0 to 7.5 m. The emission spectra were recorded from 300 to 400 nm, and the center of mass of each spectrum (νc) was calculated using the equation, νc=Σ νiFi/Σ Fi where Fi is the fluorescence emitted at wave number νi. Sample Preparation for X-ray Absorption Spectroscopy Studies—The catalytic domain of TACE was concentrated by ultra filtration using Vivaspin 6-ml units (Vivascience AG; 10-kDa cut-off) to a final concentration of 0.1 mm (2.92 mg/ml). The samples were loaded into copper sample holders (10 × 5 × 0.5 mm) precovered with Mylar tape, which is transparent to X-rays, immediately followed by freezing in liquid nitrogen. The frozen samples were then mounted inside a Diplex closed cycle helium cryostat, and their temperature was maintained at 30 K to minimize thermal disorder in the XAS data. XAS Data Collection—Data collection was performed at the National Synchrotron Light Source at Brookhaven National Laboratory, beam line X9B. The spectra were recorded at the zinc K-edge (zinc absorption edge energy in which a 15-core electron is excited by the x-ray beam) in fluorescence geometry at a low temperature (30 K). The beam energy was defined using a flat Si (111) monochromator crystal. The incident beam intensity I0 was recorded using an ionization chamber. The fluorescence intensity was recorded using a 13-element germanium detector. The transmission signal from a zinc foil was measured with a reference ion chamber simultaneously with fluorescence to calibrate the beam energy position throughout all measurements. Five or six scans of each sample were collected. The samples were checked for burning marks after each scan, and the beam position on the sample was changed before each scan to minimize radiation damage. The enzyme integrity was checked before and after exposure to X-rays. In these experiments, TACE was found to be intact and fully active. XAS Data Processing and Analysis—The average zinc K-edge absorption coefficient μ(E), was obtained after averaging 5–13 independent XAS measurements for each sample. Each data set was aligned using the first inflection point of a reference zinc metal foil (9659 eV). Subsequently, the absorption coefficients for different samples were shifted in x-ray energy until their first inflection points were aligned at the same energy. The smooth atomic background was then removed with the AUTOBK program of the UWXAFS data analysis package, developed at the University of Washington at Seattle (18Stern E.A. Newville M. Ravel B. Yacoby Y. Haskel D. Physica B. 1995; 209: 117-120Google Scholar). For background removal, the energy shift, E0, was chosen as the edge shift of the processed data for each sample and used as the origin of the photoelectron energy. The R space region for minimizing the signal below the first shell was chosen between 0.6 and 1.2 Å. Upon background removal, the useful k range (energy range used in fitting procedure, in wave units) in the resultant k2-weighted (k) was between 0 and 10.0 Å-1. Model data for fitting procedures were constructed by extracting the structural zinc site coordinates (in a radius of 4 Å from the zinc) of gelatinase A (Protein Data Bank code 1CK7) and stromelysin-1 (Protein Data Bank code 1SLM). The theoretical photoelectron scattering amplitudes and phase shifts were calculated for each zinc ligand (path), using the computer code FEFF7 (19Rehr J.J. Mustre de leon J. Zabinsky S.I. Albers R.C. J. Am. Chem. Soc. 1991; 113: 5135-5138Google Scholar, 20Zabinsky S.I. Rehr J.J. Ankudinov A. Albers R.C. Eller M.J. Phys. Rev. B. 1995; 52: 2995-2999Google Scholar). The total theoretical signal (k) was constructed by adding the most important partial k values that contributed to the R range of interest. The theoretical XAFS signal was fitted to the experimental data using the nonlinear least squares method, implemented in the program FEFFIT 2.98 (18Stern E.A. Newville M. Ravel B. Yacoby Y. Haskel D. Physica B. 1995; 209: 117-120Google Scholar) in R space, by Fourier transforming of both theoretical and experimental data. The experimental data and theoretical values were weighted by k and multiplied by a Hanning window function in Fourier transforms. The Pro Domain of TACE Can Be Expressed in Isolation—It has been shown previously that the pro domain of TACE displays inhibitory activity against this enzyme (8Milla M.E. Leesnitzer M.A. Moss M.L. Clay W.C. Carter H.L. Miller A.B. Su J.-L. Lambert M.H. Willard D.H. Sheeley D.M. Kost T.A. Burkhart W. Moyer M. Blackburn R.K. Pahel G.L. Mitchell J.L. Hoffman C.R. Becherer J.D. J. Biol. Chem. 1999; 274: 30563-30570Google Scholar). To characterize the role of TACE Pro as an enzyme inhibitor, we attempted its expression in E. coli. TACE Pro was expressed at high levels and accumulated as inclusion bodies. The purification protocol involved a nickel chelate chromatography step under denaturing conditions followed by refolding via dialysis. Refolding of TACE Pro was the limiting step in obtaining large amounts of the protein, because of its tendency to aggregate at concentrations over 8 μm. Nevertheless, the final yield for this refolding step was typically 31–48%. After refolding, TACE Pro was over 95% pure judging by reducing SDS-PAGE analysis. The protein partitioned between two forms: a free monomer and a disulfide-linked dimer (Fig. 1). The free monomer constituted 40% of the purified protein, as determined by densitometric analysis of gels ran at different total protein concentrations. TACE Pro appeared to be folded according to both our fluorescence and circular dichroism spectroscopy data. The intrinsic tryptophan fluorescence emission of TACE Pro after excitation at 280 nm showed a maximum at 325 nm, suggesting that its two tryptophan residues (Trp111 and Trp153) were in an environment of low polarity (Fig. 2A). For comparison, when the protein was subjected to chemical denaturation with 6 m Gdn-HCl, the fluorescence emission maximum shifted to the red, indicating the exposure of the tryptophans to bulk solvent in the unfolded polypeptide (Fig. 2A). The circular dichroism spectrum of TACE Pro showed a minimum between 208 and 230 nm, which revealed the presence of significant secondary structure in the protein (Fig. 2B). Upon chemical denaturation with Gdn-HCl, the CD ellipticity in this region was lost (Fig. 2B). To determine whether the pro domain of TACE is stably folded in isolation, we studied its unfolding under equilibrium conditions by monitoring the change in 1) the center of mass of the fluorescence emission and 2) the circular dichroism ellipticity at 222 nm versus denaturant concentration (Fig. 3). Both of these probes reported a single transition between the folded and unfolded states, characteristic of a cooperative, thermodynamically stable native state. The midpoint of this transition was observed at 1 m Gdn-HCl. TACE Pro Is an Inhibitor of TACE—We proceeded to test whether refolded TACE Pro could act as an inhibitor of TACE in vitro. We assayed the inhibitory activity of TACE Pro against two different TACE forms: 1) the catalytic domain of TACE alone and 2) the catalytic and disintegrin/cysteine-rich domains of TACE. TACE Pro proved to be a very potent inhibitor of the catalytic domain (IC50 = 70 nm; Fig. 4A). Interestingly, this inhibitory potency dropped over 30-fold against the form containing the catalytic and disintegrin/cysteine-rich domains (IC50 higher than 2 μm; Fig. 4A). The inhibition curve for the mature ectodomain of TACE could not be completed because TACE Pro aggregated at concentrations around 8 μm. The disintegrin/cysteine-rich domain seems to affect the ability of the pro domain to stably bind the catalytic domain. To test whether the disintegrin/cysteine-rich domain of TACE promotes dissociation of TACE Pro from the catalytic domain, we preincubated inactive pro-catalytic domain complexes with increasing amounts of disintegrin/cysteine-rich domain and then assayed for enzymatic activity. As shown in Fig. 5, even a short preincubation time (5 min) resulted in significant recovery of the catalytic activity of TACE (for example, at 10 μm of disintegrin/cysteine-rich domain, 38% of activity was observed relative to a control preparation of enzyme devoid of the pro domain; in contrast, only 3.5% of activity was observed with no addition of the disintegrin/cysteine-rich domain). Larger increases in activity were observed after 2 h of preincubation (Fig. 5; 58% activity at 10 μm disintegrin/cysteine-rich domain, versus 3.4% in its absence). Therefore, this domain of TACE appears to decrease the affinity of the pro domain for the catalytic domain. The low levels of spontaneous activation in the absence of the disintegrin/cysteine-rich domain are in agreement with previous observations showing that the catalytic domain activation of TACE was only promoted efficiently upon the addition of hydrophobic thiol-modifying reagents (8Milla M.E. Leesnitzer M.A. Moss M.L. Clay W.C. Carter H.L. Miller A.B. Su J.-L. Lambert M.H. Willard D.H. Sheeley D.M. Kost T.A. Burkhart W. Moyer M. Blackburn R.K. Pahel G.L. Mitchell J.L. Hoffman C.R. Becherer J.D. J. Biol. Chem. 1999; 274: 30563-30570Google Scholar).Fig. 5TACE activation following incubation with the disintegrin/cysteine-rich domain. 100 nm samples of inactive TACE procatalytic domain complexes were preincubated for 5 min (○) or 4 h (•) with isolated TACE disintegrin/cysteine-rich domain in 20 mm Tris-HCl, pH 8, 150 mm NaCl. After incubation, the samples were diluted and assayed for activity as described under "Materials and Methods." Activity is expressed as a percentage of control incubations lacking TACE Pro.View Large Image Figure ViewerDownload (PPT) The inhibitory activity of TACE Pro could potentially be attributed to chelation of the zinc ion in the active site of the enzyme by the C-terminal His6 purification tag in TACE Pro. We addressed this possibility by using a TACE Pro variant that lacked that purification tail. This pro variant proved to have similar inhibitor profiles when tested against both forms of TACE (data not shown). An Intact Cysteine Switch Is Not Required for Inhibition— The pro domain of TACE contains a conserved cysteine residue at position 184 in the context of a cysteine switch consensus motif. This residue was expected to mediate inhibition via coordination to the zinc ion in the active site, as proposed in the cysteine switch model (9van Wart H. Birkedal-Hansen B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5578-5581Google Scholar, 10Galazka G. Windsor L.J. Birkedal-Hansen H. Engler J.A. Biochemistry. 1996; 35: 11221-11227Google Scholar, 11Galazka G. Windsor L.J. Birkedal-Hansen H. Engler J.A. Biochemistry. 1999; 38: 1316-1322Google Scholar). To study the importance of the cysteine switch motif in the inhibition of TACE, we generated a variant of TACE Pro carrying a cysteine to alanine substitution at position 184 (TACE Pro C184A). The mutant protein was expressed in E. coli and purified using the same protocol as for TACE Pro. TACE Pro C184A purified entirely as a monomer (data not shown) and had similar solution properties as its wild type counterpart. The fluorescence emission and CD spectra of TACE Pro C184A were basically indistinguishable from those of the wild type protein (Fig. 2). TACE Pro C184A also had similar equilibrium denaturation profiles to those of TACE Pro (Fig. 3). Surprisingly, the introduction of this cysteine to alanine mutation had only a modest effect in the inhibitory potency of TACE Pro. The IC50 values obtained for TACE Pro C184A were 40 nm against the catalytic domain and higher than 2 μm against the mature ectodomain (catalytic plus disintegrin/cysteine-rich domains). This suggests that the cysteine residue cannot determine by itself the interaction between the pro and catalytic domains of TACE. Other residues within the cysteine switch region (PKVCGYLKVD190) do not appear to be critical for the pro-catalytic domain interaction either, because alanine substitutions at each individual position of the switch region, in the context of TACE Pro C184A, affected the inhibitory ability of TACE Pro modestly, within 2-fold of the IC50 observed with wild type TACE Pro (Table I). Additional sequence elements within the pro domain different from the cysteine switch region, yet to be identified, must mediate the pro-catalytic domain interaction.Table IHalf-inhibitory concentrations (IC50) for a series of alanine mutants scanning the cysteine switch region of TACE in the presence of a C184A mutation Each protein was assayed against the catalytic domain of TACE as described under "Materials and Methods." The data are the averages of two independent determinations (experimental errors within 15%) and are expressed as the nanomolar concentrations of the respective pro domain double mutant.TACE Pro mutantIC50nmP181A48.55K182A72.58V183A99.71G185A131.7Y186A70.98L187A57.32K188A87.98V189A69.24D190A81.87 Open table in a new tab Conformational Changes within the Active Site of the TACE Zymogen Probed by XAS—We wanted to address whether binding of TACE Pro to the catalytic domain of this enzyme changes the conformation and electronic state of the coordination shells surrounding the catalytic zinc ion. For this, we used x-ray absorption spectroscopy. XAS refers to modulations in x-ray absorption coefficient, μ(E), around an x-ray absorption edge of a given atom. Fig. 6A shows the XANES spectra of active and latent (bound to the pro domain) forms of TACE. The edge energy position of the pro-catalytic complex is shifted to higher energy by ∼0.45 eV in comparison with active TACE (catalytic domain alone). A shift in edge position is often an indication of structural modification at the metal site, such as different ligation (21Kleifeld O. Frenkel A. Martin J.M.L. Sagi I. Nat. Struct. Biol. 2003; 10: 98-102Google Scholar). Specifically, this shift reflects a change in the total effective charge of the zinc ion in TACE,
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