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Crystallization and preliminary X-ray analysis of porcine synovial collagenase
J. Mol. Biol. (1989) 210, 237-238
Crystallization
and Preliminary X-ray Analysis of Porcine Synovial Collagenase
Crystals of porcine synovial collagenase suitable for an X-ray structure analysis have been obtained. The crystals belong to space group 14, with unit cell dimensions a= b= 1600 A, c=53.1 A, with one molecule in the asymmetric unit. Diffraction extends beyond 3 A perpendicular to the c axis but along the 4-fold axis, the intensities are measurable only to 4a.
collagenase (M, approx. 44,000). Both enzymes were purified as described (Cawston & Tyler, 1979; Clark & Cawston, 1989) in active form in 25 m&r-sodium cacodylate (pH 7.4), 10 mM-CaCl,, 1 M-NaCl, 0.05% (w/v) Brij 35. Before crystallization experiments, the protein was concentrated using Centricon 30 miniconcentrators (Amicon) to a final protein concentration of approximately 6 mg/ml. The Brij 35 detergent was shown to have no apparent effect on crystallization by comparing results obtained with detergent present and after removal by affinity chromatography (“Extractigel” Pierce Chemicals); it was therefore routinely present t’o minimize losses of protein by adsorption to vessel walls, etc. Preliminary studies with human fibroblast collagenase were carried out, but enzyme degradation under both storage and crystallization conditions proved a major problem. A subsequent batch of the enzyme was purified using the rapid method described by Moore & Spilburg (1986). elution from the affinity column being achieved by washing with a low molecular weight inhibitor as the final step of purificat’ion. This inhibitor was also retained in the subsequent crystallization trials, and prevented the degradation previously experienced. Tiny needle crystals were obtained by microdialysis of the protein against 1.2 ivf-NaCl, 50 mM-Pipes (pH 7.5) at 4°C. However, attempts to increase the size of these their morphology crystals or change were unsuccessful. The porcine synovial collagenase proved far more amenable t#o crystallization and, in part,icular, it’ did not suffer any degradation under crystallization conditions. Poor quality crystals of porcine collagenasc were obtained at 18°C. and all subsequent experiments were set up at 4°C. Trials involving high salt conditions were unsuccessful. Crystals were obtained with polyethylene glycol as precipitant. by vapour diffusion using the hanging drop technique. The droplets initially consisted of 2 ~1 of the protein solution, at a concentration of 6.5 mg/ml in
Collagens constitute the most abundant proteins of the extracellular matrix in mammalian organisms. Collagen turnover is normally very slow. However, collagen metabolism increases dramatically in the physiological remodelling processes of growth and development, as well as in the pathology of degradative diseases such as arthritic conditions (Harris et al., 1975; Dayer et al., 1977), wound healing (Grill0 & Gross, 1967; Vaes, 1972) and tumour invasion and metastasis (Liotta et al., 1983; Salo et al., 1985). The initiation of collagen breakdown requires specific enzymes, collagenases, which catalyse the initial step in the proteolytic degradat’ion of collagen. Collagenases are zinccontaining metalloproteinases that rcquirc calcium for thermal stability (Seltzer et al., 1976). Several types of collagenases can be distinguished on the basis of their immunological properties and their substrate specificity for different types of collagen (Hibbs et al., 1985; Seltzer et al., 1981). The interstitial collagenases from human skin (Stricklin et a/., 1977: Goldberg et al., 1986), human gingiva (Wilhelm et al., 1984), pig synovium (Cawston & Tyler, 1979). rabbit bone (Hembry et al., 1986) and rabbit synovium (Vat’er et aZ., 1981) have been purified and characterized. They all cleave the native helix of interstitial collagens, types I, II and III, at a single specific Gly-Ile or Gly-Leu bond located three-fourt’hs of the distance from the N terminus (Miller et al., 1976; Hoffman et al., 1978). The regulation of these metalloproteinases is complex, involving cytokines and growth factors in their expression (Murphy & Docherty, 1988; Edwards et al., 1987), activation of then latent secreted forms (Grant et al., 1987; Cawston et aZ., 1981; Murphy et al., 1987) and inhibition of their activated forms by a specific inhibitor of metalloproteinases, TIMP, which is produced by the same cells (Murphy et a,l.. 1981: Cawston, 1986). The potential therapeutic value of low molecular weight collagenase inhibitors has been reviewed (Johnson et design of such inhibitors al., 1987). The rational would be greatly aided by detailed structural knowledge of the actjive site of collagenase from X-ray crystallographic: studies. The collagenases under study are human fibroblast collagenase (M, 42,520) and porcine synovial 00~2~d830/89/610;?:1’iO:!
$03.00/0
50 mM-Pipes
(pH
7.5) plus
2 ~1 of the
reservoir
solu-
tion that’ contained 5% (w/v) polyethylene glycol 8000 in 50 mm-Pipes buffer (pH 7.5). Some crystal polymorphism was observed and. almost invariably as the crystals grew larger, t,he central part showed disorder. A wide range of additives. over a range ot 237
% 1989 Academic Press Limited
238
L. F. Lloyd et al.
concent’rat’ions was employed, but none was successful in completely removing this partial disorder. It was found that, a slight increase in pH, using Hepes buffer (pH 7%), gave better ordered crystals, which tend to grow in pairs, of typical dimensions for a single crystal of 200 pm x 200 pm x 100 pm. Crystals grown under the different conditions described have been examined by X-ray diffraction. All the crystals obtained have the same lattice with unit cell dimensions, n =b= 161.0 8, c=FjY.l a (1 L&=O.l nm). The space group is 14 with one molecule in the asymmetric unit. The crystal volume per unit protein molecular mass, V, is 3.8 A per dalton, which indicates a very high solvent content (680/0. v/v). This is consist’ent’ with a low resistance of the crystals to mechanical damage, and with a crystal densit,y of approximately 1.11 g/cm3. The diffraction patterns extend beyond 3 A resolution perpendicular to the c axis, but along the 4-fold axis, the intensities are measurable to about 4 A. A complete native data set has been collected on a’ FAST area detector (Enraf-Nonius, Delft), and a search for heavy-atom derivatives is in progress. This work has been funded by Beecham Pharmaceut~icals Research Division and the Arthritis and Rheumatism Council. We thank the many researchers from the Arthritis Research Project, and Biological Pilot Plant of Beecham Pharmaceuticals who contributed to the supply of the collagenases.
L. F. Lloyd T. Skariyfiski A. J. Wonacott Blackett Laboratory Imperial College London SW7 2BZ, C’.K.
T. E. Cawston I. M. Clark Rheumatology Research lrnit Addenbrooke’s Hospit,al Cambridge CB2 2&Q, U.K.
C. J. Mannix G. P. Harper Beecham Pharmaceuticals Research Division Coldharbour Road Harlow. Essex, CM19 5AD. L!.K. and Yew Tree Bottom Road Epsom. Surrey KT18 5XQ. L’.K. Received 4 July 1989
References Cawston. ‘1‘. E. (1986). In f’rotpil~/~~? inhibitors (Barrtat t A. G. & Reynolds. .J. ,I.. eds). pp. 589~606. Nsevirr. Amsterda,m. (Tawston. T. E. & Tyler. .I. A: (1979). fjiochrn,. .I. 183. 647. 656. (‘awston. T. E.. Merc:c~r.E. Hr T,&r. ,I. .\. (19X1). /Go~hin/. Biophys. A eta, 657. 73%33. Clark. T. M. Pr Cawston. T. F:. (1989). Rio&en. ./ III thch p”‘“” Dayer. #J.-M.. Russell. K. C;. & Krallr, S. 11. (I!CS). Aciencp, 195, ltll-1% Edwards. I). R.. Murphy. (i.. Itr~noltls. .I. J.. II’hitham. S. E.. Docherty. A. .J. I’.. Angel. P. & Ht&h. .J. K. (1987). EMHO./. 6, 1899-1904. Goldberg. (2. I.. Wilhelm. S. M.. Kronberger. A.. Issuer. E:. A.. (*ra,nt, (:. &i. CyrI’:isen. -4. Z. (1986). .I. Hiol. C:hem. 261. 6600-6605. Grant, (:. A., Eisen. A. Z.. Jlarmer. 13. I,.. Roswit, I%‘. T. & Goldberg. (:. I. (1987). .I. Riol. (‘~PII!. 262. 5886-5889. Grille. H. C’. Br (iross. .I. (1967). I)P/Y/o~~. /2io/. 15. 300-3 17. Harris, E. T).. Jr. Faulkner. (‘. S. di HrowtI. F. E. (1!)75). (‘lin. Whop. R&t. Rus. 110. XX!-416. Hembry. R,. 11.. Murphy, (i.. (Iawston. 1‘. E.. I)inple. *I. ‘I’. & Reyrlolds, .J. J. (1986). .I. (‘r/l Xvi. 81. 105- 1% Hibbs: M., Hasty. K. A., Seyrr. .J. 11.. liang. ,. H. &I Mainardi. C’. M. (1985). .1. /Iio/. C’h~no. 260. %493-2.500. Hoffman, H.. Fiet,zrk, I’. I’. & Kuhn. K. (1!47X). .I. J/o/. Biot. 125. 197-165. ,Johnson. LV, I-1.. Roberts. N. A. B Borkakotl. S. (IHU). J. Jhzyme InhiM. 2. I 22. I,iot,ta. 1,. A.. Rae. (‘. ?;. & Barsky. S. H. (19%). /N/J. Inued 49, 636. ,Miller. E. *I., Harris. Is. I)..
Edited by A. h’luy
Crystallization
and Preliminary X-ray Analysis of Porcine Synovial Collagenase
Crystals of porcine synovial collagenase suitable for an X-ray structure analysis have been obtained. The crystals belong to space group 14, with unit cell dimensions a= b= 1600 A, c=53.1 A, with one molecule in the asymmetric unit. Diffraction extends beyond 3 A perpendicular to the c axis but along the 4-fold axis, the intensities are measurable only to 4a.
collagenase (M, approx. 44,000). Both enzymes were purified as described (Cawston & Tyler, 1979; Clark & Cawston, 1989) in active form in 25 m&r-sodium cacodylate (pH 7.4), 10 mM-CaCl,, 1 M-NaCl, 0.05% (w/v) Brij 35. Before crystallization experiments, the protein was concentrated using Centricon 30 miniconcentrators (Amicon) to a final protein concentration of approximately 6 mg/ml. The Brij 35 detergent was shown to have no apparent effect on crystallization by comparing results obtained with detergent present and after removal by affinity chromatography (“Extractigel” Pierce Chemicals); it was therefore routinely present t’o minimize losses of protein by adsorption to vessel walls, etc. Preliminary studies with human fibroblast collagenase were carried out, but enzyme degradation under both storage and crystallization conditions proved a major problem. A subsequent batch of the enzyme was purified using the rapid method described by Moore & Spilburg (1986). elution from the affinity column being achieved by washing with a low molecular weight inhibitor as the final step of purificat’ion. This inhibitor was also retained in the subsequent crystallization trials, and prevented the degradation previously experienced. Tiny needle crystals were obtained by microdialysis of the protein against 1.2 ivf-NaCl, 50 mM-Pipes (pH 7.5) at 4°C. However, attempts to increase the size of these their morphology crystals or change were unsuccessful. The porcine synovial collagenase proved far more amenable t#o crystallization and, in part,icular, it’ did not suffer any degradation under crystallization conditions. Poor quality crystals of porcine collagenasc were obtained at 18°C. and all subsequent experiments were set up at 4°C. Trials involving high salt conditions were unsuccessful. Crystals were obtained with polyethylene glycol as precipitant. by vapour diffusion using the hanging drop technique. The droplets initially consisted of 2 ~1 of the protein solution, at a concentration of 6.5 mg/ml in
Collagens constitute the most abundant proteins of the extracellular matrix in mammalian organisms. Collagen turnover is normally very slow. However, collagen metabolism increases dramatically in the physiological remodelling processes of growth and development, as well as in the pathology of degradative diseases such as arthritic conditions (Harris et al., 1975; Dayer et al., 1977), wound healing (Grill0 & Gross, 1967; Vaes, 1972) and tumour invasion and metastasis (Liotta et al., 1983; Salo et al., 1985). The initiation of collagen breakdown requires specific enzymes, collagenases, which catalyse the initial step in the proteolytic degradat’ion of collagen. Collagenases are zinccontaining metalloproteinases that rcquirc calcium for thermal stability (Seltzer et al., 1976). Several types of collagenases can be distinguished on the basis of their immunological properties and their substrate specificity for different types of collagen (Hibbs et al., 1985; Seltzer et al., 1981). The interstitial collagenases from human skin (Stricklin et a/., 1977: Goldberg et al., 1986), human gingiva (Wilhelm et al., 1984), pig synovium (Cawston & Tyler, 1979). rabbit bone (Hembry et al., 1986) and rabbit synovium (Vat’er et aZ., 1981) have been purified and characterized. They all cleave the native helix of interstitial collagens, types I, II and III, at a single specific Gly-Ile or Gly-Leu bond located three-fourt’hs of the distance from the N terminus (Miller et al., 1976; Hoffman et al., 1978). The regulation of these metalloproteinases is complex, involving cytokines and growth factors in their expression (Murphy & Docherty, 1988; Edwards et al., 1987), activation of then latent secreted forms (Grant et al., 1987; Cawston et aZ., 1981; Murphy et al., 1987) and inhibition of their activated forms by a specific inhibitor of metalloproteinases, TIMP, which is produced by the same cells (Murphy et a,l.. 1981: Cawston, 1986). The potential therapeutic value of low molecular weight collagenase inhibitors has been reviewed (Johnson et design of such inhibitors al., 1987). The rational would be greatly aided by detailed structural knowledge of the actjive site of collagenase from X-ray crystallographic: studies. The collagenases under study are human fibroblast collagenase (M, 42,520) and porcine synovial 00~2~d830/89/610;?:1’iO:!
$03.00/0
50 mM-Pipes
(pH
7.5) plus
2 ~1 of the
reservoir
solu-
tion that’ contained 5% (w/v) polyethylene glycol 8000 in 50 mm-Pipes buffer (pH 7.5). Some crystal polymorphism was observed and. almost invariably as the crystals grew larger, t,he central part showed disorder. A wide range of additives. over a range ot 237
% 1989 Academic Press Limited
238
L. F. Lloyd et al.
concent’rat’ions was employed, but none was successful in completely removing this partial disorder. It was found that, a slight increase in pH, using Hepes buffer (pH 7%), gave better ordered crystals, which tend to grow in pairs, of typical dimensions for a single crystal of 200 pm x 200 pm x 100 pm. Crystals grown under the different conditions described have been examined by X-ray diffraction. All the crystals obtained have the same lattice with unit cell dimensions, n =b= 161.0 8, c=FjY.l a (1 L&=O.l nm). The space group is 14 with one molecule in the asymmetric unit. The crystal volume per unit protein molecular mass, V, is 3.8 A per dalton, which indicates a very high solvent content (680/0. v/v). This is consist’ent’ with a low resistance of the crystals to mechanical damage, and with a crystal densit,y of approximately 1.11 g/cm3. The diffraction patterns extend beyond 3 A resolution perpendicular to the c axis, but along the 4-fold axis, the intensities are measurable to about 4 A. A complete native data set has been collected on a’ FAST area detector (Enraf-Nonius, Delft), and a search for heavy-atom derivatives is in progress. This work has been funded by Beecham Pharmaceut~icals Research Division and the Arthritis and Rheumatism Council. We thank the many researchers from the Arthritis Research Project, and Biological Pilot Plant of Beecham Pharmaceuticals who contributed to the supply of the collagenases.
L. F. Lloyd T. Skariyfiski A. J. Wonacott Blackett Laboratory Imperial College London SW7 2BZ, C’.K.
T. E. Cawston I. M. Clark Rheumatology Research lrnit Addenbrooke’s Hospit,al Cambridge CB2 2&Q, U.K.
C. J. Mannix G. P. Harper Beecham Pharmaceuticals Research Division Coldharbour Road Harlow. Essex, CM19 5AD. L!.K. and Yew Tree Bottom Road Epsom. Surrey KT18 5XQ. L’.K. Received 4 July 1989
References Cawston. ‘1‘. E. (1986). In f’rotpil~/~~? inhibitors (Barrtat t A. G. & Reynolds. .J. ,I.. eds). pp. 589~606. Nsevirr. Amsterda,m. (Tawston. T. E. & Tyler. .I. A: (1979). fjiochrn,. .I. 183. 647. 656. (‘awston. T. E.. Merc:c~r.E. Hr T,&r. ,I. .\. (19X1). /Go~hin/. Biophys. A eta, 657. 73%33. Clark. T. M. Pr Cawston. T. F:. (1989). Rio&en. ./ III thch p”‘“” Dayer. #J.-M.. Russell. K. C;. & Krallr, S. 11. (I!CS). Aciencp, 195, ltll-1% Edwards. I). R.. Murphy. (i.. Itr~noltls. .I. J.. II’hitham. S. E.. Docherty. A. .J. I’.. Angel. P. & Ht&h. .J. K. (1987). EMHO./. 6, 1899-1904. Goldberg. (2. I.. Wilhelm. S. M.. Kronberger. A.. Issuer. E:. A.. (*ra,nt, (:. &i. CyrI’:isen. -4. Z. (1986). .I. Hiol. C:hem. 261. 6600-6605. Grant, (:. A., Eisen. A. Z.. Jlarmer. 13. I,.. Roswit, I%‘. T. & Goldberg. (:. I. (1987). .I. Riol. (‘~PII!. 262. 5886-5889. Grille. H. C’. Br (iross. .I. (1967). I)P/Y/o~~. /2io/. 15. 300-3 17. Harris, E. T).. Jr. Faulkner. (‘. S. di HrowtI. F. E. (1!)75). (‘lin. Whop. R&t. Rus. 110. XX!-416. Hembry. R,. 11.. Murphy, (i.. (Iawston. 1‘. E.. I)inple. *I. ‘I’. & Reyrlolds, .J. J. (1986). .I. (‘r/l Xvi. 81. 105- 1% Hibbs: M., Hasty. K. A., Seyrr. .J. 11.. liang. ,. H. &I Mainardi. C’. M. (1985). .1. /Iio/. C’h~no. 260. %493-2.500. Hoffman, H.. Fiet,zrk, I’. I’. & Kuhn. K. (1!47X). .I. J/o/. Biot. 125. 197-165. ,Johnson. LV, I-1.. Roberts. N. A. B Borkakotl. S. (IHU). J. Jhzyme InhiM. 2. I 22. I,iot,ta. 1,. A.. Rae. (‘. ?;. & Barsky. S. H. (19%). /N/J. Inued 49, 636. ,Miller. E. *I., Harris. Is. I)..
Edited by A. h’luy