J. Mol. Biol.

(1991)

222,

865-868

Crystals of the Complex between Human Growth Hormone and the Extracellular Domain of Its Receptor Mark Ultsch, Abraham

M. de Vast and Anthony

A. Kossiakoff

Department of Protein Engineering Genentech, Inc., 460 Point San Bruno Boulevard South San Francisco, CA 94080, U.S.A. (Received

12 August

1991; accepted 30 August

1991)

Single crystals suitable for high-resolution diffraction studies have been grown of the human growth hormone (hGH) complexed to the extracellular domain of its cloned receptor from the human liver (hGHbp), using the technique of repeat seeding. The crystals are in space group P2,2,2, with a = 1458 A, b = 68.6 A, c = 76-O A, and diffract to at least 27 A resolution on a rotating anode X-ray source. Analysis of the composition of these crystals showed the stoichiometry of the complex to be hGH : (hGHbp),. This finding, coupled with biochemical data on the complex in solution, indicates that the biologically significant dimerization of the growth hormone receptor is mediated through a single hormone molecule. Structure determination of the complex is currently being completed. Keywords:

crystallization;

human growth hormone; stoichiometry

Human growth hormone (hGH$), a 191 amino acid residue protein, is synthesized in the anterior pituitary. Its role in the promotion of growth, coupled with changes in the metabolism of proteins, carbohydrates and lipids, is well documented (Paladini et al., 1983). The hGH gene has been cloned and expressed in Escherichia co& both as the methionyl derivative (Goeddel et al., 1979) and as the naturally occurring sequence without an amino-terminal methionine (Chang et al., 1987). The human growth hormone receptor (Leung et al., 1987) has a single extracellular domain (28 kD), a transmembrane segment, and an intracellular domain (30 kD) that is not homologous to any known tyrosine kinase or other protein. The extracellular portion of the hGH receptor is structurally related to the extracellular domain of the prolactin receptor (Boutin et al., 1988) and broadly to at least eight other cytokine and related receptors (Cosman et al., 1990; Bazan, 1990; Patthy, 1990). The extracellular domain (residues 1 to 246, of the hGH receptor occurs naturally in serum as an hGH binding protein. A slightly truncated version of the binding protein,

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complex;

residues 1 to 238, was expressed in E. coli (Fuh et al., 1990) and used in our crystallization studies. This truncated form (hGHbp) retains the same specificity and high affinity for hGH as the natural binding protein found in serum (Ku = 64 nM; Fuh et al., 1990). The mechanism(s) whereby external signals from polypeptide hormones are transduced through the membrane by their receptors into intracellular messages is fundamental to our understanding of molecular endocrinology. Hormone-induced oligomerization has been proposed for a large family of tyrosine kinase receptors (Yarden & Ullrich, 1988; Ullrich & Schlessinger, 1990), and for receptors of EGF (Schechter et al., 1979; Schreiber et al., 1983; Yarden & Schlessinger, 1987a,b), insulin (Kahn et al., 1978; Heffetz & Zick, 1986; Kubar & Van Obberghen, 1989), PDGF (Heldin et al., 1989; Hammacher et aZ., 1989; Seifert et al., 1989), and IGF-1 (Ikari et al., 1988). Crystals have been grown of the complex between IL-2 and the a-subunit of its receptor (Lambert et al., 1989), and of an EGFreceptor complex (Gunther et al., 1990). However, in neither case was hormone-induced oligomerization of the receptor reported. Recently, we have found that crystals of the complex between hGH and hGHbp contain a 1 : 2 mixture of hormone/receptor; moreover, this stoichiometry of the complex was demonstrated to exist in solution as well (B. C. Cunningham, M. Ultsch, A. M. De Vos, M. G.

t Author to whom all correspondence should be addressed. 1 Abbreviations used: hGH, human growth hormone. MPD, 2-methyl,2,4-pentanediol; h.p.l.c., high-pressure liquid chromatography; PMSF, phenylmethylsulfonyl fluoride. OO22-2836/91/240865Xb4

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Figure 1. Phenylsuperose f.p.1.c. (Pharmacia) profile of hGHbp. Frozen cell paste was thawed in hypotonic buffer (10 miw-Tris, pH 8.0, 1 m&r-PMSF, 25 mM-EDTA). The suspension was homogenized, stirred for 1 h at 4”C, and centrifuged at 10,000 g for 20 min. Solid ammonium sulfate was added to the supernatant at 260 g/l, and the mixture was stirred until the salt had dissolved. The protein precipitate was collected by centrifugation at 10,000 g for 30 min. The pellet was resuspended in 10 mw-Tris, 1 mM-PMSF (pH 80), and dialyzed against the same buffer. The dialyzate was loaded onto an hGH affinity column (controlled glass pore (Sigma)). After washing, the column was eluted with 2 M-KSCN, 20 m&r-Tris (pH 75), 1 miw-PMSF, 25 mM-EDTA. The peak fraction was dialyzed against 10 mM-Tris (pH 7-5), 1 mm-PMSF. The hGHbp was loaded onto a phenylsuperose f.p.1.c. column (Pharmacia) and eluted isocratitally in a stepwise fashion (above). Fractions were assayed by SDS/polyacrylamide gel electrophoresis, and intact hGHbp was separated from a clipped form. Intact hGHbp was incubated overnight at 4°C with /lZ macroglobulin, loaded on a Mono Q f.p.1.c. column (Pharmacia). and eluted with a linear gradient of 0 to 0.2 M-NaCl. Peaks were assayed by SDS/polyacrylamide gel electrophoresis, and intact hGHbp was pooled and desalted using a PDlO column (Pharmacia) into 50 mu-sodium acetate, 1 mM-PMSF (pH 55).

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Figure 2. Purification of the complex on a size exclusion column. After overnight incubation of hGH and hGHbp at 4°C: the solution was loaded onto a (:75-120 Sephadex size exclusion column (Sigma), equilibrated with 120 mivi-NaCl. 20 mmsodium acetate 1 mivr-PMSF (pH 5.5). The complex fractions were pooled, concentrated and desalted in 50 mi%-sodium acetate. 1 miw-PMSF (pH 5.5).

adapted from Fuh et al. (1990) (see the legend to Fig. 1). For the production of crystals suitable for X-ray diffraction studies, it was critical to remove any clipped forms of the hGHbp and to prevent subsequent clipping from occurring. This was accomplished by adding fi2 macroglobulin (Boehr-

inger-Mannheim) at 1 pg/mg hGHbp in order to remove any traces of protease. To form the hormone-receptor complex, hGHbp was added to hGH and allowed to incubate for 24 hours at 4°C. Initially. a 1 : 1 ratio of hGH/hGHbp was used, but after the stoichiometry of the complex (hGH : (hGHbp),, below) had been established. a 1 : 2 ratio was used. X-ray diffraction quality crystals only grew when excess components were removed from the crystallization solution, and this was accomplished on a size exclusion column (Fig. 2). The concentration of the complex in the

final stock solution was about 4 mg/ml (based on ~“‘“(280 nm) = 1.67). Mulkerrin & J. A. Wells, unpublished results). Here, we report the purification of the hGHbp and the crystallization of the complex. (a)

hGHbp (residues E. coli as described purification process

Purijkation 1 to 238) was expressed in by Fuh et al. (1990). The for recombinant hGHbp was

(b) Crystallography Crystals of the hGH-recept.or complex were grown using a combination of vapor diffusion in sitting drops along with a repeat seeding procedure. Initially, crystals were obtained using the hanging drop method. The reservoir contained 40% (w/v) saturated ammonium sulfate, 1 oo MPD. and the

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‘W 15

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Figure 3. A 15” precession photograph of the h01 zone of the crystals of the hGH-receptor complex. Precession photographs were taken on an Enraf-Nonius camera, mounted on a Rigaku RU200 rotating anode generator, operated at 45 kV, 110 mA. The edge of the picture corresponds to a resolution of 3 A. Three-dimensional data sets were collected to a resolution of 2.8 A.

crystallization drop was a mixture of 10 ~1 of complex (4 mg/ml) in Tris (pH 7*5), and 2 ~1 of the reservoir. Small crystals grew within a week; however, in order to grow diffraction quality crystals, repeat seeding was necessary. The stock solution of complex was diluted to 1.7 mg/ml using 0.1 M-bis-Tris (pH 65), to which saturated ammonium sulfate was added to make a 10% saturated solution, and MPD to a final concentration of 1%. The mixture (50 ~1) was allowed to equilibriate for two days at room temperature, at which time seed crystals were introduced. Within two weeks, crystals suitable for X-ray diffraction studies were obtained with dimensions of 1 mmx04mm x0.1 mm. Precession photography (Fig. 3) was used to establish the space group as P2,2,2, with unit cell dimensions of a = 145.8 A, b = 68.8 A, c = 76-O A (1 A = 0.1 nm). These crystals diffract to at least 2.7 A resolution using a rotating anode X-ray source. The composition of the crystals was analyzed using a variety of techniques, including gel filtration, electrophoresis and high-pressure liquid chromatography (h.p.1.c.). The ratio of hGH/ hGHbp was quantified by monitoring absorbance at 214 nm of their respective peaks separated on h.p.1.c. (Fig. 4). Comparison of peak areas to those of standard solutions containing known ratios of hGH : hGHbp indicated the stoichiometry of the complex to be 1 hGH : 2 hGHbp. Integration of peak areas for four independent determinations gave a ratio of A214 of the hGH peak to the hGHbp

1, 25

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Figure 4. Composition analysis of the crystals of the hGH-receptor complex. (a) to (c) High-pressure liquid chromatograms of standard solutions with hGH : hGHbp ratios of 1 : 1, 1 : 2 and 1 : 3, respectively. (d) Crystals were washed in mother liquor and dissolved in @l y0 (v/v) trifluoroacetic acid, and chromatographed under denaturing conditions. The amount of hGH to hGHbp was quantified by monitoring their respective peaks at 214 nm, which corresponds to the absorbance of peptide bonds. For a 1: 2 hGH : hGHbp complex, the expected ratio for the integrated peaks is @40, based on 190 peptide bonds in hGH, and 237 in the hGHbp.

peak of 042 f0.02. For a complex having a 1 : 2 ratio of hGH : hGHbp, the predicted ratio for the integrated peak areas is @40, compared to a ratio of @SO for a 1 : 1 stoichiometry. Thus, our crystals contain a complex of the form hGH : (hGHbp),. That this stoichiometry is not an artifact of crystallization was subsequently confirmed by analysis of the complex in solution (B. C. Cunningham et al., unpublished results). This finding has important implications for the biological mechanism of action of growth hormone, and possibly of a group of relaxed cytokines such as interleukins 2, 3, 4 and 6, and granulocyte-macrophage stimulating factors. In the case of hGH, where two identical receptors are bound by a molecule without any apparent duplication in sequence or structure, the receptors must bind to sites having quite different surface characteristics. The structure of the complex should provide the stereochemical details of the two receptor-hormone interfaces, as well as any possible receptor-receptor interactions. Data collection on our crystals was done on an Enraf-Nonius “FAST” area detector mounted on a Rigaku, RUZOO rotating anode generator. Native data sets have been collected to a resolution of 2.8 il, and two heavy-atom derivatives have been found. We are currently completing the structure determination of the complex.

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We thank Ken Olson for samples of hGH, Brad Snedecor and Mike Covarrubias for fermentation, Mike Mulkerrin for useful suggestions on purification, Jim Wells for discussions, and Bill Henzel for amino acid analyses.

References Bazan, J. F. (1990). Structural design and molecular evolution of a cytokine receptor superfamily. Proc. Nat.

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Boutin, J.-M., Jolicoeur, C., Okamura, H., Gagnon, J.. Edery, M., Shirota, M., Banville, D., Dusanter-Fourt, I., Djiane. J. & Kelly, P. A. (1988). Cloning and expression of the rat prolactin receptor, a member of the growth hormone/prolactin receptor gene family. Cell, 53, 69-77. Chang, C. N., Rey, M., Bochner, B., Heyneker, H. & Gray, G. (1987). High-level secretion of human growth hormone by Escherichia coli. Gene (Amsterdam), 55, 1899196. Cosman, D., Lyman, 6. D., Idzerda, R. L., Beckmann, M. P., Park, L. S., Goodwin, R. G. & March, C. J. (1990). A new cytokine receptor superfamily. Trends Biochem. Sci. 15, 265-270. Fuh, G., Mulkerrin, M. G., Bass, S., McFarland, N., Brochier, M., Bourell, J. H., Light. D. R. & Wells, J. A. (1990). The human growth hormone receptor. Secretion from Escherichia coli and disulfide bonding pattern of the extracellular binding domain. J. Biol. Chem. 265, 3111-3115. Goeddel, D. V., Heyneker, H. L., Hozumi, T., Arentzen. R., Itakura, K., Yansura, D. G., Ross, M. J.. Miozzari, G., Crea, R. & Seeburg, P. H. (1979). Direct expression in Eeeherichia coli of a DNA sequence coding for human growth hormone. Nature (London), 281, 544-548. Gunther, N., Betzel, C. & Weber. W. (1990). The secreted form of the epidermal growth factor receptor. Characterization and crystallization of the receptor-ligand complex. J. Biol. Chem. 265, 22082-22085. Hammacher, A., Mellstrom, K., Heldin, C.-H. & Westermark, B. (1989). Isoform-specific induction of actin reorganization by platelet-derived growth factor suggests that the functionally active receptor is a dimer. EMBO J. 8, 2489-2495. Heffetz, D. & Zick, Y. (1986). Receptor aggregation is necessary for activation of the soluble insulin receptor kinase. J. Biol. Chem. 261, 889-894. Heldin, C-H., Ernlund, A., Rorsman, C. & Riinnstrand, L. (1989). Dimerization of the B-type plateletderived growth factor receptors occurs after ligand binding and is closely associated with receptor kinase activation. J. Biol. Chem. 264, 89058912. Ikari, N., Yoshino, H., Moses, A. C. & Flier, J. S. (1988). Evidence that receptor aggregation may play a role

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Edited by A. Klug