Crystallization of trimeric recombinant human tumor necrosis factor (cachectin).

Crystals of tumor necrosis factor (TNF) have been obtained in two forms. Rhombohedral crystals grow in 1.8 to 2.0 M ammonium sulfite, pH 7.8 at 21 degrees C, and tetragonal crystals grow in 2.6 M magnesium sulfate, pH 5.5 at 25 degrees C. Analysis of TNF by isoelectric focusing under native and denaturing conditions indicates that TNF molecules exist as trimers in solution. The rhombohedral cachectin crystals belong to space group R3 and have unit cell constants a = b = c = 47.65 A and alpha = beta = gamma = 88.1 degrees. Density determinations and the space group indicate that the unit cell contains one 51,000-dalton trimer. These crystals are stable in the x-ray beam and diffract to at least 1.85 A but are apparently twinned by merohedry. The tetragonal crystals are space group P4(3)2(1)2 or its enantiomorph P4(1)2(1)2 and have unit cell constants a = b = 95.08, c = 117.49. The asymmetric unit contains one trimer; the crystals are stable in the x-ray beam and diffract to beyond 3 A.

117.49. The asymmetric unit contains one trimer; the crystals are stable in the x-ray beam and diffract to beyond 3 A.
Tumor necrosis factor (cachectin) is a metabolically active protein secreted by macrophages in response to certain invasive stimuli, particularly in response to challenge with endotoxin. It is believed to be an important mediator of many infection-related phenomena including induction of fever, shock, and cachexia (see Ref. 1 for review). TNF' is present at elevated levels in the serum of Mycobacterium bouis strain BCG-infected mice treated with endotoxin, and passive immunization of mice against TNF can prevent endotoxininduced death (2). Further, the hormone has been demonstrated to have specific cytolytic effects against certain tumorigenic cells. TNF has been demonstrated to bind with high affinity to an as yet uncharacterized cellular receptor.
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The sequence of mature TNF is 28% identical to that of human lymphotoxin, a related lymphokine that interacts with the same class of receptors (8).
TNF has been isolated, purified, and characterized (3). The mature protein (157 residues, M, = 17,000) is derived by cleavage of the 76 amino-terminal residues from the prohormone, contains two cysteines believed to form an intrachain disulfide bond, and is not glycosylated. Under native conditions TNF has been reported to exist in oligomeric states with molecular weights ranging from 34,000 to 140,000. Recent reports, however, indicate that the hormone is active as a trimer (4).
In this report we describe the conditions for growth of crystals suitable for x-ray crystallographic structure determination and describe experiments that confirm the trimeric quaternary structure of TNF in solution.

MATERIALS AND METHODS
TNF was produced from a synthetic construct, which was expressed as an intracellular protein in yeast. The protein was purified as described previously (5) and maintained in storage buffer (0.02 M Tris, pH 8.0, 0.15 M NaC1) at a concentration of 20 mg/ml. Analysis by SDS-PAGE and isoelectric focusing were performed on a Pharmacia PhastSystemTM unit using standard separation and development techniques. PhastGelTM gradient 10-15% gels were used for SDS-PAGE, and IEF 5-8 gels were used for the IEF separations. Two-dimensional, one dimension denatured isoelectric focusing (ODD-IEF) was run with the first dimension under native conditions on a standard IEF 5-8 gel. The sample lane (approximately 5 mm wide) was then cut out, inverted, and applied across the cathodal end of the second dimension gel, a PhastGel IEF 3-9, which had been soaked for 20 min in 5% Pharmalyte 3-10,6 M urea, and 0.1% Triton X-100. The gel was then run under standard conditions for an IEF 3-9 gel and developed as above.
Chromatofocusing was performed on a Pharmacia LKB Biotechnology Inc. FPLCTM system using the Mono-PTM HR 5/20 column pre-equilibrated with 25 mM BisTris, pH 6.7. TNF was applied in the equilibration buffer at a concentration of 0.75 mg/ml and eluted at a rate of 0.5 ml/min with a solution containing 10 ml of Polybuffer 74TM/100 ml of H20, pH 5.0. Buffer exchange and protein concentration were performed in a Centricon-loTM concentration device.
Rhombohedral crystals were grown at 22 "C in hanging drops equilibrated by vapor diffusion. Typically, 5 pl of the protein in storage buffer was combined on a siliconized coverslip with an equal volume of a reservoir solution containing 200 mM BES and 1.8-2.0 M ammonium sulfite at pH 7.8. The coverslip was then inverted over 1 ml of the reservoir solution contained in the well of a Linbro cell culture plate and sealed with vacuum grease. Macroseeding was performed by introducing a thoroughly washed crystal (0.1 mm on edge) into a 15-pl hanging drop containing 5 p1 of 3 M BES buffered ammonium sulfite and 10 pl of protein concentrated to 33 mg/ml. Tetragonal crystals were grown as above but with a reservoir solution containing 200 mM MES and 2.4-2.7 M magnesium sulfate at pH 5.5.
Crystal density determinations were performed in a Ficoll 400 gradient essentially as described by Westbrook (6). Partial specific volume was calculated (7) from the amino acid sequence (8). Crystals were mounted in thin-walled glass capillary tubes, and precession photographs were taken using CuK, radiation from a Rigaku Rotaflex rotating anode generator at 50 kV and 108 mA. Data to 3 8, from the rhombohedral crystals and 4.5 A from the tetragonal crystals were collected by 0.75 "C w peak scans at 0.5 deg/min and 15-5 background counts at room temperature with a Rigaku Rotaflex generator operating at 5.4 kilowatts on a Rigaku AFC-5R diffractometer.

RESULTS AND DISCUSSION
Characterization-Purified TNF migrates as a single sharp band with M , = 17,000 (Fig. 1, lane A ) on SDS-polyacrylamide gels. However, isoelectric focusing under native conditions reveals a series of four banding groups each containing from one to four sub-bands with PI values between 6.7 and 5.8 (Fig.   1, lune D). The band groups are uniformly separated, consistent with groups of species differing by integral or constant electrical charge. In the hope that a more homogeneous protein sample would improve crystal size and quality, we attempted to isolate single bands by chromatofocusing. The elution profile of the Mono-P column contained four peaks with no discernible substructure which were shown by IEF to correspond to groups I-IV. An in vitro TNF cytotoxicity assay (9) demonstrated that all fractions were equally active. Fractions containing pure band group IV TNF were pooled and concentrated to 20 mg/ml.
The source of charge heterogeneity has not been determined. SDS-PAGE analysis of band groups I-IV separated by chromatofocusing shows no evidence of degradation of the 17-kDa monomer. Amino-terminal peptide sequences obtained by automated Edman degradation (13) for both unfractionated TNF and band group IV TNF indicate the lass of the NH2-terminal valine residue (8) from roughly 50% of the molecules? Loss of the amino-terminal valine residue is unlikely to generate significant change in the PI of the subunit. Elman analysis (10) of the unfractionated protein indicated that no reduced thiols were present, thus ruling out heterogeneity in the oxidation state of cysteine residues. It is possible that deamidation of one or more of the 16 asparagine or glutamine residues is responsible for the heterogeneity of recombinant TNF. Isoelectric focusing of TNF in the presence of 6 M urea, which would favor dissociation of oligomers, reveals three major bands (Fig. 1, lune I) Fig. 1, lane I. The four bands of group IV dissociate in the second dimension into bands a and b. Band groups I1 and I11 both dissociate into a, b, and c, but group I1 contains proportionately more species c than group 111. Band group I contains only species c. As discussed in the text, this pattern suggests that the molecule exists as a trimer under native conditions. Proposed monomer composition of the major bands in the native gel are shown in C.
comes markedly heterogeneous (Fig. 1, lune E ) after storage in sample buffer at 4 "C for 2 months (as opposed to the unfractionated sample, which was maintained at -70 "C). The seven band groups visible in this lane are similar in subbanding pattern and band separation to the four band groups in the fresh TNF. Isoelectric focusing under denaturing conditions reveals additional monomeric species which are either absent or present only as minor contaminants in fresh TNF samples (data not shown).
The complex banding pattern produced by fresh purified TNF (or samples stored at -70 "C) in IEF gels run under native conditions (Fig. 1, lune D) is proposed to arise from trimers containing different combinations of the three major (and several minor) monomeric species. Substructure within individual band groups is expected because two or more combinations could yield trimeric species with similar PI values.
The trimeric structure of TNF is deduced from two-dimensional ODD-IEF gels (Fig. 2) described above by identifying the monomeric species that comprise the individual band groups in the native IEF gel. The observed pattern of banding is consistent with a trimeric, but not a dimeric, molecule. The four constituents of band group IV migrate in the native dimension (Fig. 2) with a mean PI of 6.6. These dissociate in the denaturing dimension into two monomeric species, a and b, with closely spaced PI values which fall within the range of group IV oligomers. The combination of two monomer species into four unique oligomeric species is expected for trimers; the four bands on the native dimension correspond to the four possible combinations of the two monomers (aaa, aab, abb, bbb). Dimerization of the a and b monomers, on the other hand, would produce only three bands in the native gel. Likely monomer compositions are indicated for the remaining major bands in Fig. 2. Our results are in agreement with recently reported results from neutron-scattering experiments which indicate that TNF is a trimer in solution (11) and with cross-linking studies that indicate that TNF is active as a trimer (4).
Crystallization-We have obtained crystals of cachectin in two space groups. Rhombohedral crystals (Fig. 3B) are produced when ammonium sulfite is used as a precipitant while tetragonal crystals have been grown from magnesium sulfate. Rhombohedral crystals were obtained with the unfractionated protein in ammonium sulfite at concentrations ranging from 1.8 to 2.0 M at a pH of approximately 7.8 at 21 "C. The crystals are highly birefringent and rhombohedral in shape. Crystals first appear after 36 h and grow to 0.1 mm on edge in a few days. Rhombohedra as large as 0.22 mm on edge have been grown by seeding with the 0.1-mm crystals. Crystallization attempts with ammonium sulfate have been entirely unsuccessful, even in trials containing 10 mM ammonium sulfite. Also, attempts with ammonium sulfate and either pmercaptoethanol or dithiothreitol as a reducing agent have been unproductive. SDS-PAGE (Fig. 1, lune B ) and IEF (Fig.  1, lane H ) of washed, redissolved rhombohedral crystals indicate that the lattice contains primarily band group I protein, which is present in barely detectable quantities in the original crystallization solution. This may explain our lack of success with the purified band group IV TNF; crystallization attempts with this fraction resulted in showers of microcrystals. Analysis of precession photographs and diffractometer data indicate a rhombohedral unit cell with a = b = c = 47.65 A, and a = / 3 = y = 88.1", space group R3. By using measured density (1.22 g/cc) and calculated partial specific volume (0.74 cc/g), the number of molecules per unit cell was calculated to be 2.55, not inconsistent with a trimer in the unit cell, considering possible experimental error in density measurements. For a trimer, the computed Matthew's coefficient, V,, is 2.12 A/dalton, well within the range of reported values (12). The reciprocal lattice shows approximate RTM symmetry (consistent with space group R32); however, the extent of 2-fold symmetry about the a* and b* axes varies from crystal to crystal, suggesting that the pseudo-symmetry is due to twinning by merohedry (14). R32 symmetry implies six molecules per unit cell, resulting in a? unreasonable value for the packing density ( V , of 1.08 A3/dalton). Twinning fractions (15) estimated from data sets for selected crystals range from 35 to 45%. Despite the observed twinning, the crystals are well formed and highly birefringent. Measurements from 15min still photographs and preliminary data collection trials indicate that the crystals diffract to at least 1.85 A, are stable to a monochromatized x-ray beam, and show a loss of only 10% in intensity of three low order reflections after 70 h of irradiation at room temperature. All attempts to grow untwinned crystals in this space group, including alteration of pH, precipitant, and crystal growth rate, have been unsuccessful.
Tetragonal crystals of TNF were obtained with the unfractionated protein in 2.6 M magnesium sulfate at pH 5.5. Crystal growth is quite temperature-dependent; the largest single crystals are grown when the crystallization plate is kept at approximately 20 "C for the first 48 h and then shifted to 25 "C. Crystals typically appear after 72 h and grow to 0.2 X 0.2 X 0.8 mm in several days. Analysis of precession photographs and diffractometer data indicate that the crystals belong to space group P 4~2~2 or its enantiqmorph P41212 aad have unit cell dimensions a = b = 95.08 A and c = 117.5 A.
The crystals diffract to at least 3.0 A and are stable in the xray beam. A search for heavy atom derivatiyes is in progress. The computed packing density (V, = 2.6 A/dalton) is consistent with the presence of three monomers in the asymmetric unit or 24 monomers per unit tell. A self-rotation function (16) computed with a native 4.5 A resolution data set reveals a local 3-fold axis of symmetry parallel to the a* and b* axes, indicating the presence of trimers of TNF in the asymmetric unit. The two crystal forms of TNF reported here and those reported by other groups (11,17) all contain multiples of three TNF molecules in the unit cell, suggesting that TNF is a trimer in the crystalline state as well as in solution.