J. MoZ. Biol. (1990) 211, 683-684
Crystals of the NC1 Domain of Human Type IV Collagen Milton Stubbs, Lesley Summers, Irmgard Mayr, Monika Schneider Wolfram Bode, Robert Huber Abteilung
Strukturforschung, Max-Plan& Institut fiir Biochemie D-8033 Martinsried bei Miinchen, F. R.G.
Albert Ries and Klaus Kiihn Abteilung
Bindegewebsforschung, D-8033 Martinsried
Max-Plan& Institute ftir bei Miinchen, F.R.G.
Biochemie
(Received 9 November 1989; accepted 15 November 1989) Crystals obtained
of the non-collagenous C-terminal region (NCl) of type IV collagen have been from human placenta. These crystals diffract to 2.0 8, and belong to space group P22,2,, with cell dimensions a=81 A, b=158& c=138& cc=B=y=90”. The crystals contain one hexamer in the asymmetric unit; they are very stable with respect to X-rays.
Type IV collagen is the principal protein component of basement membranes. It differs from the fibre-forming collagen types I, II and III in that the molecules form a two-dimensional network rather than unidimensional rods (Timpl et al., 1981). Monomeric type IV collagen is a heterotrimer of two a,(IV) chains and one cr,(IV) chain (Kiihn, 1986). It consists of a 390 nm long triple helical domain, frequently interrupted by non-helical sections, and terminates in a globule at the C terminus, the NC1 domain. In the complex macromolecular network, collagen IV molecules are covalently linked by their like ends, four via the triple-helical N termini and two via the C-terminal NC1 domains (Timpl et al., 1981). In addition, the molecules are laterally associated at the triple-helical regions in an as yet unknown manner (Yurchenco & Ruben, 1987). Here. we describe crystals of the hexameric NC1 complex. formed through association of the C t,ermini of two collagen IV molecules. The primary structures of the cr(IV) NC1 domains of mouse, human and Drosophila., deduced from cDNA sequences, is highly conserved (Oberbkumer et al.. 1985; Schwarz-Magdolen et al., 1986; Kurkinen et al., 1987: Pihlajaniemi et al., 1985; Brinker et aZ., 1985; Hostikka et al., 1987: Cecchini et al., 1987; Blumberg et al., 1987). The two a-chains show 97% homology between human and mouse in the NC1 region, and there is a 59% homology between the cl-chain of Drosophila and the mammalian u-chains. In contrast to within the triple-helical regions of type IV collagen, the a,(IV) and a2(IV) chains show a high degree (65%) of homology in the NC1 0022%2836/90/040683~2
$03.00/O
683
domain. Each NC1 domain, about 220 amino acid residues in length, consists of two homologous (35%) sub-domains, each with a set, of six cysteine residues. Identical patterns of intrachain disulphide bridges are formed within each subunit. giving the entire domain a four-leaved clover-like arrangement. During dimerization of two collagen TV molecules, a highly ordered hexameric complex of four a,(IV)NCl and two a,(IV)NCl domains is formed, which is subsequently stabilized by intermolecular disulphide bridges (Siebold et al.: 1988). The disulphide exchange occurs mainly between the a,(IV)NCl domains; a2(IV)NCl dimers have been much observed to a lesser extent, and a,(IV)-a,(IV)NCl heterodimers have not been detected (Weber et aZ., 1984). Dimeriza’trion is a prerequisite for hexamer formation (Webrr et al., 1988). The three-dimensional structure of this highly symmetric molecule is important in our attempts to understand the structural requirements for the aggregation of the NC1 domains and for the formation of the intermolecular disulphides. Large prismshaped crystals of mouse tumour NCI, suitable for high-resolution X-ray analysis, have been reported (Timpl et al.! 1985). The monoclinic crystals, obtained from phosphate containing 15 o/o polyethylene glycol at pH 8.5, belong to space group P2, with cell dimensions a=740A, b=158,!, c=139A, a=y=90” and /?=98” (1 A =O.l nm). The difficulties encountered in trying to reproduce t,he crystals, coupled with an asymmetric unit of two 0
1990 Academic
Press Limited
684
M. Stubbs et’ al.
hexamers, prompted the search for a new crystal form. NC1 hexamers were isolated from human placenta and purified as described (Weber et al., 1984). The lyophilized protein was dissolved in 5 mM-ammonium bicarbonate at a concentration of 10 mg/ml. The protein was crystallized by the vapour diffusion method in hanging drops. Crystals were obtained at room temperature using reservoirs containing 64 to 65 iv-sodium citrate buffer at pH 70. Each drop contained 45 ~1 of protein solution and .1.5 ~1 of reservoir solution. Sometimes, drops were seeded with small plate-like crystals after 24 hours, then left to grow for up to three months. Maximal dimensions observed were 1.0 mm x 0.7 mm x 0.2 mm. The crystals diffract to 2.0 A resolution, and show remarkable resilience to X-ray exposure. A complete native dataset has been collected using the FAST television area detector mounted on a Rigaku rotating anode X-ray source, operated with the package (Pflugrath & MADNES software Messerschmidt, 1985; Messerschmidt & Pflugrath, 1987). The crystals belong to space group P22,2,, with unit cell dimensions a=81 A, b=158 A, a=j?=y=90”. The unconventional c=138A, crystal setting was chosen to mark the clear homology with the monoclinic mouse form mentioned earlier. A local 2-fold axis of the monoclinic crystal (either within 1 or between 2 hexamers) now coincides with the new crystallographic a axis. With one hexamer per asymmetric unit, a packing density V,= 2.65 A per dalton is obtained, well within the range described for protein crystals (Matthews, 1968). Three other crystal forms were observed for human NCl, using either citrate or phosphate buffers containing 15% polyethylene glycol at a range of pH values between 6 and 10. They were, for high-resolution X-ray however, unsuitable analysis. The search for heavy-metal derivatives is underway. Edited
References B.. MacKreIl, A. J., Olson, P. F.. Blumberg, Kurkinen, M., Monson, J. M., Katzle, J. E. & Fessler, J. H. (1987). J. Biol. Chem. 262, 5947-5950. Brinker, J. M., Gudas, L. J., Loidl, H. R., Wang, S.-Y., Rosenbloom, J., Kefalides, N. A. & Myers, cJ.C. (1985). Proc. Nat. Acad. Sci., U.S.A. 82, 3649-3653. Cecchini, J.-P., Knibiehler, B., Mirre, C. & Le Parco, Y. (1987). Eur. J. Biochem. 165, 587-593. Hostikka, S. L., Kurkinen. M. & Tryggvason, K. (1987). FEBS Letters, 216, 281-286. Kuhn, K. (1986). Rheumatology, 10, 2C-29. Kurkinen, M., Condon, M. R., Blumberg. B., Barlow, D. P., Quinones, S., Sas, J. & Pihlajaniemi, T. (1987). .I. Biol. Chem. 262, 849668499. Matthews, B. W. (1968). J. Mol. Biol. 33, 491-497. Messerschmidt. A. & Pflugrath. J. W. (1987). J. Appl. Crystallogr. 20, 306315. Oberbaumer, 1.. Laurent, M.. Schwarz, U., Sakurai, Y.. Vogeli. G., voss. T.. Siebold, B., Yamada, Y., Glanville, R. W. & Kuhn. K. (1985). Eur. J. Biochem. 147, 217-224. Pflugrath. J. W. & Messerschmidt, A. (1985). Crystallography in Molecular Biology. Meeting Abstracts, Bischenberg, France. Pihlajaniemi. T., Tryggvason, K., Myers, ,J. C.. Kurkinen, M., Lebo, R., Cheung, M. C., Prockop, D. & Boyd C. D. (1985). J. Biol. Chem. 260, 7681-7687.
Schwarz-Magdolen, U., Oberbaumer, I. & Kiihn.
K.
(1986). FEBS Letters, 208, 2033207. Siebold, B.; Deutzmann. R. & Kuhn, K. (1988). Eur. J. Biochem. 176. 617-624. Timpl, R., Wiedemann, H., van Delden, V., Furthmayr, H. & Kuhn, K. (1981). Eur. J. B&hem. 120. 203-211. Timpl, R., Oberbaumer, I., von der Mark, H., Bode, W., Wick, G.. Weber. S. & Engel, J. (1985). In Biology, Collagen Pathology of and Chemistry (Fleischmajer, R., Olsen, B. R. & Kuhn, K., eds), vol. 460, pp. 58-72, New York Academy of Science. New York. Weber, S., Engel, J., Wiedemann. H., Glanville, R. W. & Timpl, R. (1984). Eur. J. Biochem. 139, 401410. Weber, S., Diilz, R., Timpl, R., Fessler. J. H. & Engel, *J. (1988). Eur. J. Biochem. 175, 229-236. Yurchenco, P. D. & Ruben, G. C. (1987). J. C’ell. Biol. 105, 2559-2568. by A. Klug
Crystals of the NC1 Domain of Human Type IV Collagen Milton Stubbs, Lesley Summers, Irmgard Mayr, Monika Schneider Wolfram Bode, Robert Huber Abteilung
Strukturforschung, Max-Plan& Institut fiir Biochemie D-8033 Martinsried bei Miinchen, F. R.G.
Albert Ries and Klaus Kiihn Abteilung
Bindegewebsforschung, D-8033 Martinsried
Max-Plan& Institute ftir bei Miinchen, F.R.G.
Biochemie
(Received 9 November 1989; accepted 15 November 1989) Crystals obtained
of the non-collagenous C-terminal region (NCl) of type IV collagen have been from human placenta. These crystals diffract to 2.0 8, and belong to space group P22,2,, with cell dimensions a=81 A, b=158& c=138& cc=B=y=90”. The crystals contain one hexamer in the asymmetric unit; they are very stable with respect to X-rays.
Type IV collagen is the principal protein component of basement membranes. It differs from the fibre-forming collagen types I, II and III in that the molecules form a two-dimensional network rather than unidimensional rods (Timpl et al., 1981). Monomeric type IV collagen is a heterotrimer of two a,(IV) chains and one cr,(IV) chain (Kiihn, 1986). It consists of a 390 nm long triple helical domain, frequently interrupted by non-helical sections, and terminates in a globule at the C terminus, the NC1 domain. In the complex macromolecular network, collagen IV molecules are covalently linked by their like ends, four via the triple-helical N termini and two via the C-terminal NC1 domains (Timpl et al., 1981). In addition, the molecules are laterally associated at the triple-helical regions in an as yet unknown manner (Yurchenco & Ruben, 1987). Here. we describe crystals of the hexameric NC1 complex. formed through association of the C t,ermini of two collagen IV molecules. The primary structures of the cr(IV) NC1 domains of mouse, human and Drosophila., deduced from cDNA sequences, is highly conserved (Oberbkumer et al.. 1985; Schwarz-Magdolen et al., 1986; Kurkinen et al., 1987: Pihlajaniemi et al., 1985; Brinker et aZ., 1985; Hostikka et al., 1987: Cecchini et al., 1987; Blumberg et al., 1987). The two a-chains show 97% homology between human and mouse in the NC1 region, and there is a 59% homology between the cl-chain of Drosophila and the mammalian u-chains. In contrast to within the triple-helical regions of type IV collagen, the a,(IV) and a2(IV) chains show a high degree (65%) of homology in the NC1 0022%2836/90/040683~2
$03.00/O
683
domain. Each NC1 domain, about 220 amino acid residues in length, consists of two homologous (35%) sub-domains, each with a set, of six cysteine residues. Identical patterns of intrachain disulphide bridges are formed within each subunit. giving the entire domain a four-leaved clover-like arrangement. During dimerization of two collagen TV molecules, a highly ordered hexameric complex of four a,(IV)NCl and two a,(IV)NCl domains is formed, which is subsequently stabilized by intermolecular disulphide bridges (Siebold et al.: 1988). The disulphide exchange occurs mainly between the a,(IV)NCl domains; a2(IV)NCl dimers have been much observed to a lesser extent, and a,(IV)-a,(IV)NCl heterodimers have not been detected (Weber et aZ., 1984). Dimeriza’trion is a prerequisite for hexamer formation (Webrr et al., 1988). The three-dimensional structure of this highly symmetric molecule is important in our attempts to understand the structural requirements for the aggregation of the NC1 domains and for the formation of the intermolecular disulphides. Large prismshaped crystals of mouse tumour NCI, suitable for high-resolution X-ray analysis, have been reported (Timpl et al.! 1985). The monoclinic crystals, obtained from phosphate containing 15 o/o polyethylene glycol at pH 8.5, belong to space group P2, with cell dimensions a=740A, b=158,!, c=139A, a=y=90” and /?=98” (1 A =O.l nm). The difficulties encountered in trying to reproduce t,he crystals, coupled with an asymmetric unit of two 0
1990 Academic
Press Limited
684
M. Stubbs et’ al.
hexamers, prompted the search for a new crystal form. NC1 hexamers were isolated from human placenta and purified as described (Weber et al., 1984). The lyophilized protein was dissolved in 5 mM-ammonium bicarbonate at a concentration of 10 mg/ml. The protein was crystallized by the vapour diffusion method in hanging drops. Crystals were obtained at room temperature using reservoirs containing 64 to 65 iv-sodium citrate buffer at pH 70. Each drop contained 45 ~1 of protein solution and .1.5 ~1 of reservoir solution. Sometimes, drops were seeded with small plate-like crystals after 24 hours, then left to grow for up to three months. Maximal dimensions observed were 1.0 mm x 0.7 mm x 0.2 mm. The crystals diffract to 2.0 A resolution, and show remarkable resilience to X-ray exposure. A complete native dataset has been collected using the FAST television area detector mounted on a Rigaku rotating anode X-ray source, operated with the package (Pflugrath & MADNES software Messerschmidt, 1985; Messerschmidt & Pflugrath, 1987). The crystals belong to space group P22,2,, with unit cell dimensions a=81 A, b=158 A, a=j?=y=90”. The unconventional c=138A, crystal setting was chosen to mark the clear homology with the monoclinic mouse form mentioned earlier. A local 2-fold axis of the monoclinic crystal (either within 1 or between 2 hexamers) now coincides with the new crystallographic a axis. With one hexamer per asymmetric unit, a packing density V,= 2.65 A per dalton is obtained, well within the range described for protein crystals (Matthews, 1968). Three other crystal forms were observed for human NCl, using either citrate or phosphate buffers containing 15% polyethylene glycol at a range of pH values between 6 and 10. They were, for high-resolution X-ray however, unsuitable analysis. The search for heavy-metal derivatives is underway. Edited
References B.. MacKreIl, A. J., Olson, P. F.. Blumberg, Kurkinen, M., Monson, J. M., Katzle, J. E. & Fessler, J. H. (1987). J. Biol. Chem. 262, 5947-5950. Brinker, J. M., Gudas, L. J., Loidl, H. R., Wang, S.-Y., Rosenbloom, J., Kefalides, N. A. & Myers, cJ.C. (1985). Proc. Nat. Acad. Sci., U.S.A. 82, 3649-3653. Cecchini, J.-P., Knibiehler, B., Mirre, C. & Le Parco, Y. (1987). Eur. J. Biochem. 165, 587-593. Hostikka, S. L., Kurkinen. M. & Tryggvason, K. (1987). FEBS Letters, 216, 281-286. Kuhn, K. (1986). Rheumatology, 10, 2C-29. Kurkinen, M., Condon, M. R., Blumberg. B., Barlow, D. P., Quinones, S., Sas, J. & Pihlajaniemi, T. (1987). .I. Biol. Chem. 262, 849668499. Matthews, B. W. (1968). J. Mol. Biol. 33, 491-497. Messerschmidt. A. & Pflugrath. J. W. (1987). J. Appl. Crystallogr. 20, 306315. Oberbaumer, 1.. Laurent, M.. Schwarz, U., Sakurai, Y.. Vogeli. G., voss. T.. Siebold, B., Yamada, Y., Glanville, R. W. & Kuhn. K. (1985). Eur. J. Biochem. 147, 217-224. Pflugrath. J. W. & Messerschmidt, A. (1985). Crystallography in Molecular Biology. Meeting Abstracts, Bischenberg, France. Pihlajaniemi. T., Tryggvason, K., Myers, ,J. C.. Kurkinen, M., Lebo, R., Cheung, M. C., Prockop, D. & Boyd C. D. (1985). J. Biol. Chem. 260, 7681-7687.
Schwarz-Magdolen, U., Oberbaumer, I. & Kiihn.
K.
(1986). FEBS Letters, 208, 2033207. Siebold, B.; Deutzmann. R. & Kuhn, K. (1988). Eur. J. Biochem. 176. 617-624. Timpl, R., Wiedemann, H., van Delden, V., Furthmayr, H. & Kuhn, K. (1981). Eur. J. B&hem. 120. 203-211. Timpl, R., Oberbaumer, I., von der Mark, H., Bode, W., Wick, G.. Weber. S. & Engel, J. (1985). In Biology, Collagen Pathology of and Chemistry (Fleischmajer, R., Olsen, B. R. & Kuhn, K., eds), vol. 460, pp. 58-72, New York Academy of Science. New York. Weber, S., Engel, J., Wiedemann. H., Glanville, R. W. & Timpl, R. (1984). Eur. J. Biochem. 139, 401410. Weber, S., Diilz, R., Timpl, R., Fessler. J. H. & Engel, *J. (1988). Eur. J. Biochem. 175, 229-236. Yurchenco, P. D. & Ruben, G. C. (1987). J. C’ell. Biol. 105, 2559-2568. by A. Klug
