Vol. 178, No. 2, 1991 July 31, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 634-640
CRYSTALLIZATION AND PRELIMINARY STUDIES OF RECOMBINANT RABBIT
X-RAY DIFFRACTION INTERFERON-GAMMA
Cleopas T. Samudzi, Cynthia L. Gribskov, L.E. Burton+ and J. Ronald Rubin NCI-Frederick Cancer Researchand Development Center, ABL-Basic ResearchProgram, P.O. Box B, Frederick, MD 21702 *Department of ProcessDevelopment, GenentechInc., 460 Point San Bruno Blvd., South San Francisco, CA 94010 Received
May
31,
1991
Two different crystal forms of recombinant rabbit EN-y were obtained under different crystallization conditions. The first, a tetragonal form with spacegroup P4,2,2 or P4,2,2, was obtainedthrough vapor phaseequilibration using the sitting drop rods techniquewith ammonium citrate as the major precipitating agent. The unit cell dimensionsof this crystal form are a = b = 82.1 A and c = 116.3 A. These crystals diffract to 2.8 8, resolution and contain a dimer in the asymmetric unit. A second crystal form was obtained by the batch method at pH 8.0 using sodium chloride as the precipitating agent. The crystals are hexagonal, spacegroup P6,22 or P6,22, and with unit cell dimensionsof a = b = 58.0 8, and c = 169 A. This form contains monomer in the asymmetric unit and diffracts to greater than 2.7 A resolution. Both forms appearto be eminently suitablefor further analysesand crystal structure solution. 0 1991 Academic
Press,
Inc.
Interferon gamma (IFN-y), also known as immune or type II interferon, was first identified by Wheelock asa potent antiviral agent (1). This interferon is distinguishedfrom type I interferons, IFN-a, and IFN-p, by a number of factors. In particular, IFN-y is inactivated at pH 2.0, whereasEN-a and IF’N-p are not. In addition, IFN-y is antigenically distinct from type I interferons and has a lower molecular weight (17,147 daltons). It also differs from IFN-a and IFN-p in its amino acid sequence,chromosomallocation and occurrence of introns in the gene encodingIFN-y, its immunoregulatory and enhancedantiproliferative effects, aswell asthe types of substancesthat causeinduction and the cell types that produce IFN-y. The cDNA for human IFN-y was first isolated and characterized in 1981 using mRNA isolated from cultures of mitogen-stimulatedlymphocytes (2). The cDNA isolatedencodesa polypeptide 166 amino acid residuesin length. The 23 N-terminal residuesare characteristicof a eukaryotic signal sequence. The coding sequencefor the mature human IFN-y encodesa 144 residue polypeptide. This sequencewas cloned in Escherichia coli using an expressionvector containing the E. coli trp promoter. This system expresseshigh levels of soluble IFN-y protein, which specifically 0006-291x/91 $1.50 Copyrighr 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
634
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178,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
produces antiviral activity in human cells and is inactivated by antibodies against natural IFN-y. The amino acid sequence of human IFNy Differential
glycosylation
contains two potential N-linked
glycosylation
sites.
at these sites accounts for the variation in molecular weights observed
in naturally occurring IFN-y.
The biologically
active form of IFN-y is a dimer (3), and strong
denaturants such as sodium dodecyl sulfate are required to dissociate the EN-y dimers. IFN-y genes from mice, cows, rabbits, and rats have been isolated by screening genomic DNA h-libraries
with the human EN-y
cDNA
sequence (4).
A comparison of the primary
sequence of several of these IFN-y proteins shows a high degree of homology.
Sequence
identities range from 87% for the rat and mouse sequences to 39% for the human and rat sequences. Recombinant activity.
IFN-y
molecules, like their natural counterparts,
have potent antiviral
For example, recombinant murine lFN-y has been shown in viva to protect mice from
infection by encephalomyocarditisvirus (5). IFN-y exhibits antiproliferative activity both in viva (6) and in mousetumor models in viva
The most important biological role of EN-y is as an
immunoregulatory agent. EN-y induces both class I and classII (I-LA-DR) antigens of the major histocompatibility complex, and is also directly involved in macrophageactivation. This molecule is a lymphokine that can regulate the expressionand activity of other lymphokines. It alsoinducesmacrophageto secreteinterleukin-1 (7) and stimulatesthe production interleukin-2 and lymphotoxin (8). IFN-y shows synergism in antiviral activity with IFN-a and IFN$ and in antitumor activity with lymphotoxin and tumor necrosisfactor (9). Early reports suggestedthat IFN-y was a protein with a molecular weight of 40,000 to 60,000 daltons. It was later shown (10) that natural IFN-y could be resolved into two bands of molecular weight 20,000 and 25,000 daltons on SDS-polyacrylamide gels and that the early estimateswere due to that fact the IFN-y readily dimerizes. Amino acid sequenceanalysis of naturally occurring human IFN-y identified the N-terminal residue as pyroglutamate. The Cterminal end of natural IFN-y is variable due to limited proteolysis of the molecule. Deletion of 12 residuesfrom the C terminus of recombinant EN-y hasno effect on its biological activity and reducesthe tendency of IFN-y to form high-molecular-weight aggregates(11). IFN-y forms an extremely tight dimer, and dissociationof the dimer requires treatment with strong denaturantssuchasguanidine-HCI or SDS, or reduction of pH below 2.0. Following denaturation at pH 2.0 and dilution to pH 6.0, the moleculeslowly renaturesand dimerizes with a halftime of dimerization of about 8 hours as monitored by tryptophan fluorescence. Circular dichroism measurementsof the IFN-y dimer at pH 6.0 suggestthat the molecule contains a high proportion (> 70%) of a-helical secondary structure. Statistical secondary-structureprediction methods(12) have been applied to the primary sequencesof 1FN-y from several species. The predicted secondarystructurescontain a high proportion (-60%) of a-helical structure and almost 635
Vol.
178,
No.
2, 1991
no p-sheet structure. hydrophobic
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Analyses of the IFN-y amino acid sequence for periodic distribution
and hydrophilic
of
side chains also indicate the presence of amphipathic u-helical
segments (13). All of the secondary-structure
prediction methods applied to the IFN-y sequence
predict an a-helical protein consisting of five or six a-helical segments connected by relatively short turns and with little or no p-structure. In this report, we describe the crystallization
and preliminary x-ray diffraction
analysis
of the recombinant rabbit IFN-y. METHODS Highly purified samples of recombinant rabbit IFN-y were prepared as described previously (4). Two crystal forms of this interferon were obtained under different crystallization conditions. A fractional factorial approach (14) was used to survey conditions suitable for crystallization. The method used to bring about supersaturation was the vapor phase equilibration using sitting drop rods (Perpetual Systems Inc., Rockville, MU) in Linbro trays. Crystals that were large enough for diffraction data collection were grown from lo-@ droplets. Each droplet contained 5 pl of 30 mg/ml rabbit IFN-y, 20 mM sodium succinate (pH 6.0) and 5 ul of the reservoir solution containing 200 mM ammonium sulfate, 200 mM ammonium chloride, 400 mM ammonium citrate and 400 mM ammonium acetate were allowed to equilibrate at 12°C against a OS-ml solution of the reservoir. Rod-shaped crystals of rabbit IFN-y formed after about 1 week. These crystals are referred to as Type I. A second crystal form of rabbit IFN-y was obtained by batch methods using sodium chloride as the precipitating agent. Rabbit IFN-y solutions containing 4.0 mg/ml IFN-y, 40 mM Tris-HCl @H S.O), and 1.0 M NaCl (total volume = 1.5 ml) were placed in plastic centrifuge tubes and allowed to stand at 4°C. After several weeks, large hexagonal bipyramidal crystals of rabbit IFN-y formed in the tubes. These crystals are referred to as Type II. X-ray diffraction data were collected on a Siemans multiwire area detector mounted on a Rigaku RU200 x-ray generator operating at 50 mA and 100 kV. These data were collected in 0.25” frames at a rate of 360 seconds/frame and a crystal-to-detector distance of 150 mm. Highly redundant data from both Type I and Type II native crystals were collected from single crystals by measuring a total of 800 frames/crystal. RESULTS
AND DISCUSSION
The Type I crystals grew to maximum dimensions of 2.0 x 0.3 x 0.2 mm (Figure 1 top panel). Precession photographs on these crystals indicate that they are tetragonal, space group P4J2,2 or P4,2,2, with unit cell dimensions a = b = 82.10 A and c = 116.33 8, The volume of the unit cell is 784,000 A3. Assuming eight IFN-y dimers of molecular weight 34,220 in the unit cell (one dimer per asymmetric unit), the V, for these crystals is 2.86 A3/dalton.
This is within
the range expected for protein crystals (15) and corresponds to a fractional solvent volume (VJ of 57% in the unit cell. Figure 1 (bottom panel) shows the hexagonal bipyramidal Type II crystal.
They grow to
maximum dimensions of 0.5 x 0.4 x 0.3 mm. Precession X-ray photographs of these crystals (Figure 2) indicate that they are hexagonal, space group P6,22 or P6,22, with
unit cell
dimensions a = b = 57.8 A and c = 169.2 A. The volume (V) of the unit cell is 523,500 A3. 636
Vol.
178, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
w A typical tetragonal crystal of the Type I form (top panel). These rod-shaped crystals grew to maximum dimensions of 2.0 x 0.3 x 0.2 mm in about one week. The bottom panel shows the hexagonal bipyramidal crystals of the Type II form. These crystals grew to maximum dimensions of about 0.5 x 0.4 x 0.3 mm.
637
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Figure A Precession photographs of the Type II (hexagonal) form showing the hk0 zone (top panel) andI the h01 zone (bottom panel). The crystal-to-film distance was 100 mr n and the ;ion angle p = 1.5”.
638
Vol.
178,
No.
Assuming
2, 1991
BIOCHEMICAL
that there are 12 IFNy
A3/dalton (15).
AND
BIOPHYSICAL
molecules (MW
RESEARCH
COMMUNICATIONS
17,110) in the unit cell, the V, is 2.55
The protein density in this cell is higher than in the tetragonal (Type I) form.
The fractional volume of solvent in the hexagonal (Type II) form is approximately 52%. There is a single IFN-y monomer in the asymmetric
unit of structure.
This requires that the two
polypeptide chains in the rabbit IFN-y dimer have identical conformations related by a true crystallographic
and that they are
dyad axis.
Screening of potential heavy atom derivatives of the Type II crystals on the area detector is currently underway.
Suitable derivatives
solutions of K,PtCl, and K,HgI,.
have been obtained by soaking these crystals in
We are currently in the process of determining the Type II
crystal structure by the multiple isomorphous replacement technique.
ACKNOWLEDGMENTS This research was sponsored by the National Cancer Institute, DHHS, under contract no. NOl-CO-74101
with ABL-Basic
Research Program.
The contents of this publication do not
necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. REFERENCES 1. Wheelock, E.F. (1980)
Science 149, 310-311.
2. Gray, P.W., Leung, D.W., Pennica, D., Yelverton, E., Najarian, R., Simonson, C., Derynck, R., Sherwood, P.J., Wallace, D.M., Berger, S-L., Levinson, A.D. and Goeddel, D.V. (1982) Nature 295, 503-508. 3. Le, J., Barrowclough,
B.S. and Vileek, J. (1984)
J. Immunol. Methods 69, 61-70.
4. Rinderknecht, E. and Burton, L.E. (1985) In The Biology of the Interferon System (H. Kirchner, H. Schellekens, Eds.) pp. 397-402. Elsevier, Amsterdam. 5. Shalaby, M.R., Week, P.K., Rinderknecht, (1984) Cell Immunol. 81, 380-392.
E., Harkins, R.N., Frane, J.W. and Ross, M.J.
6. Czarniecki, C.W., Fennie, C.W., Powers, D.B. and Estell, D.A. (1984)
J. Virol. 49, 490-496.
7. Palladino, M.A., Sredersky, L.P., Shepard, H.M., Pearlstein, K.T., Vilcek, J. and Scheid, M.P. (1983) In Interferon Research: Clinical Application and Regulatory Consideration (K.C. Zoon, Ed.) pp. 139-147. Elsevier, Amsterdam. 8. Svredersky, L.D., Nedwin, G.E., Goeddel, D.V. and Palladino, M.A. (1985) 134. 1604-1608.
J. Immunol.
9. Stone-Wolff, D.S., Yip, Y.K., Kelker, H.C., Le, J., Henriksen-DeStefano, D., Rubin, B.Y., J. Exp. Med. 159, 828-843. Rinderknecht, E., Aggarwal, B.B. and Vilcek, J. (1984) 639
Vol.
178, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
10. Yip, Y.K., Barrowclough, B.S., Urban, C. and Vilcek, J. (1982) Science 215, 411-413. 11. Burton, L.E. Gray, P.W., Goeddel, D.V. and Rinderknecht, E. (1985) In Biology of the Interferon System, (H. Kirchner, H. Schellekens,Eds.) pp. 403-409. Elsevier, Amsterdam. 12. Chou, P.Y. and Fasman,G.D. (1974)
Biochemistry 13, 211-222.
13. Finer-Moore, J., Bazan, J.F., Rubin, J.R. and Stroud, R.M. (1989) In Prediction of Protein Structure and the Principles of Protein Conformation (G.D. Fasman,Ed.) pp. 719-759. Plenum, New York. 14. Baldwin, E.T. (1990) Ph.D. Dissertation, University of North Carolina at Chapel Hill. 15. Matthews, B.W. (1968)
J. Mol. Biol. 33, 491-497.
640
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 634-640
CRYSTALLIZATION AND PRELIMINARY STUDIES OF RECOMBINANT RABBIT
X-RAY DIFFRACTION INTERFERON-GAMMA
Cleopas T. Samudzi, Cynthia L. Gribskov, L.E. Burton+ and J. Ronald Rubin NCI-Frederick Cancer Researchand Development Center, ABL-Basic ResearchProgram, P.O. Box B, Frederick, MD 21702 *Department of ProcessDevelopment, GenentechInc., 460 Point San Bruno Blvd., South San Francisco, CA 94010 Received
May
31,
1991
Two different crystal forms of recombinant rabbit EN-y were obtained under different crystallization conditions. The first, a tetragonal form with spacegroup P4,2,2 or P4,2,2, was obtainedthrough vapor phaseequilibration using the sitting drop rods techniquewith ammonium citrate as the major precipitating agent. The unit cell dimensionsof this crystal form are a = b = 82.1 A and c = 116.3 A. These crystals diffract to 2.8 8, resolution and contain a dimer in the asymmetric unit. A second crystal form was obtained by the batch method at pH 8.0 using sodium chloride as the precipitating agent. The crystals are hexagonal, spacegroup P6,22 or P6,22, and with unit cell dimensionsof a = b = 58.0 8, and c = 169 A. This form contains monomer in the asymmetric unit and diffracts to greater than 2.7 A resolution. Both forms appearto be eminently suitablefor further analysesand crystal structure solution. 0 1991 Academic
Press,
Inc.
Interferon gamma (IFN-y), also known as immune or type II interferon, was first identified by Wheelock asa potent antiviral agent (1). This interferon is distinguishedfrom type I interferons, IFN-a, and IFN-p, by a number of factors. In particular, IFN-y is inactivated at pH 2.0, whereasEN-a and IF’N-p are not. In addition, IFN-y is antigenically distinct from type I interferons and has a lower molecular weight (17,147 daltons). It also differs from IFN-a and IFN-p in its amino acid sequence,chromosomallocation and occurrence of introns in the gene encodingIFN-y, its immunoregulatory and enhancedantiproliferative effects, aswell asthe types of substancesthat causeinduction and the cell types that produce IFN-y. The cDNA for human IFN-y was first isolated and characterized in 1981 using mRNA isolated from cultures of mitogen-stimulatedlymphocytes (2). The cDNA isolatedencodesa polypeptide 166 amino acid residuesin length. The 23 N-terminal residuesare characteristicof a eukaryotic signal sequence. The coding sequencefor the mature human IFN-y encodesa 144 residue polypeptide. This sequencewas cloned in Escherichia coli using an expressionvector containing the E. coli trp promoter. This system expresseshigh levels of soluble IFN-y protein, which specifically 0006-291x/91 $1.50 Copyrighr 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
634
Vol.
178,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
produces antiviral activity in human cells and is inactivated by antibodies against natural IFN-y. The amino acid sequence of human IFNy Differential
glycosylation
contains two potential N-linked
glycosylation
sites.
at these sites accounts for the variation in molecular weights observed
in naturally occurring IFN-y.
The biologically
active form of IFN-y is a dimer (3), and strong
denaturants such as sodium dodecyl sulfate are required to dissociate the EN-y dimers. IFN-y genes from mice, cows, rabbits, and rats have been isolated by screening genomic DNA h-libraries
with the human EN-y
cDNA
sequence (4).
A comparison of the primary
sequence of several of these IFN-y proteins shows a high degree of homology.
Sequence
identities range from 87% for the rat and mouse sequences to 39% for the human and rat sequences. Recombinant activity.
IFN-y
molecules, like their natural counterparts,
have potent antiviral
For example, recombinant murine lFN-y has been shown in viva to protect mice from
infection by encephalomyocarditisvirus (5). IFN-y exhibits antiproliferative activity both in viva (6) and in mousetumor models in viva
The most important biological role of EN-y is as an
immunoregulatory agent. EN-y induces both class I and classII (I-LA-DR) antigens of the major histocompatibility complex, and is also directly involved in macrophageactivation. This molecule is a lymphokine that can regulate the expressionand activity of other lymphokines. It alsoinducesmacrophageto secreteinterleukin-1 (7) and stimulatesthe production interleukin-2 and lymphotoxin (8). IFN-y shows synergism in antiviral activity with IFN-a and IFN$ and in antitumor activity with lymphotoxin and tumor necrosisfactor (9). Early reports suggestedthat IFN-y was a protein with a molecular weight of 40,000 to 60,000 daltons. It was later shown (10) that natural IFN-y could be resolved into two bands of molecular weight 20,000 and 25,000 daltons on SDS-polyacrylamide gels and that the early estimateswere due to that fact the IFN-y readily dimerizes. Amino acid sequenceanalysis of naturally occurring human IFN-y identified the N-terminal residue as pyroglutamate. The Cterminal end of natural IFN-y is variable due to limited proteolysis of the molecule. Deletion of 12 residuesfrom the C terminus of recombinant EN-y hasno effect on its biological activity and reducesthe tendency of IFN-y to form high-molecular-weight aggregates(11). IFN-y forms an extremely tight dimer, and dissociationof the dimer requires treatment with strong denaturantssuchasguanidine-HCI or SDS, or reduction of pH below 2.0. Following denaturation at pH 2.0 and dilution to pH 6.0, the moleculeslowly renaturesand dimerizes with a halftime of dimerization of about 8 hours as monitored by tryptophan fluorescence. Circular dichroism measurementsof the IFN-y dimer at pH 6.0 suggestthat the molecule contains a high proportion (> 70%) of a-helical secondary structure. Statistical secondary-structureprediction methods(12) have been applied to the primary sequencesof 1FN-y from several species. The predicted secondarystructurescontain a high proportion (-60%) of a-helical structure and almost 635
Vol.
178,
No.
2, 1991
no p-sheet structure. hydrophobic
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Analyses of the IFN-y amino acid sequence for periodic distribution
and hydrophilic
of
side chains also indicate the presence of amphipathic u-helical
segments (13). All of the secondary-structure
prediction methods applied to the IFN-y sequence
predict an a-helical protein consisting of five or six a-helical segments connected by relatively short turns and with little or no p-structure. In this report, we describe the crystallization
and preliminary x-ray diffraction
analysis
of the recombinant rabbit IFN-y. METHODS Highly purified samples of recombinant rabbit IFN-y were prepared as described previously (4). Two crystal forms of this interferon were obtained under different crystallization conditions. A fractional factorial approach (14) was used to survey conditions suitable for crystallization. The method used to bring about supersaturation was the vapor phase equilibration using sitting drop rods (Perpetual Systems Inc., Rockville, MU) in Linbro trays. Crystals that were large enough for diffraction data collection were grown from lo-@ droplets. Each droplet contained 5 pl of 30 mg/ml rabbit IFN-y, 20 mM sodium succinate (pH 6.0) and 5 ul of the reservoir solution containing 200 mM ammonium sulfate, 200 mM ammonium chloride, 400 mM ammonium citrate and 400 mM ammonium acetate were allowed to equilibrate at 12°C against a OS-ml solution of the reservoir. Rod-shaped crystals of rabbit IFN-y formed after about 1 week. These crystals are referred to as Type I. A second crystal form of rabbit IFN-y was obtained by batch methods using sodium chloride as the precipitating agent. Rabbit IFN-y solutions containing 4.0 mg/ml IFN-y, 40 mM Tris-HCl @H S.O), and 1.0 M NaCl (total volume = 1.5 ml) were placed in plastic centrifuge tubes and allowed to stand at 4°C. After several weeks, large hexagonal bipyramidal crystals of rabbit IFN-y formed in the tubes. These crystals are referred to as Type II. X-ray diffraction data were collected on a Siemans multiwire area detector mounted on a Rigaku RU200 x-ray generator operating at 50 mA and 100 kV. These data were collected in 0.25” frames at a rate of 360 seconds/frame and a crystal-to-detector distance of 150 mm. Highly redundant data from both Type I and Type II native crystals were collected from single crystals by measuring a total of 800 frames/crystal. RESULTS
AND DISCUSSION
The Type I crystals grew to maximum dimensions of 2.0 x 0.3 x 0.2 mm (Figure 1 top panel). Precession photographs on these crystals indicate that they are tetragonal, space group P4J2,2 or P4,2,2, with unit cell dimensions a = b = 82.10 A and c = 116.33 8, The volume of the unit cell is 784,000 A3. Assuming eight IFN-y dimers of molecular weight 34,220 in the unit cell (one dimer per asymmetric unit), the V, for these crystals is 2.86 A3/dalton.
This is within
the range expected for protein crystals (15) and corresponds to a fractional solvent volume (VJ of 57% in the unit cell. Figure 1 (bottom panel) shows the hexagonal bipyramidal Type II crystal.
They grow to
maximum dimensions of 0.5 x 0.4 x 0.3 mm. Precession X-ray photographs of these crystals (Figure 2) indicate that they are hexagonal, space group P6,22 or P6,22, with
unit cell
dimensions a = b = 57.8 A and c = 169.2 A. The volume (V) of the unit cell is 523,500 A3. 636
Vol.
178, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
w A typical tetragonal crystal of the Type I form (top panel). These rod-shaped crystals grew to maximum dimensions of 2.0 x 0.3 x 0.2 mm in about one week. The bottom panel shows the hexagonal bipyramidal crystals of the Type II form. These crystals grew to maximum dimensions of about 0.5 x 0.4 x 0.3 mm.
637
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No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
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Figure A Precession photographs of the Type II (hexagonal) form showing the hk0 zone (top panel) andI the h01 zone (bottom panel). The crystal-to-film distance was 100 mr n and the ;ion angle p = 1.5”.
638
Vol.
178,
No.
Assuming
2, 1991
BIOCHEMICAL
that there are 12 IFNy
A3/dalton (15).
AND
BIOPHYSICAL
molecules (MW
RESEARCH
COMMUNICATIONS
17,110) in the unit cell, the V, is 2.55
The protein density in this cell is higher than in the tetragonal (Type I) form.
The fractional volume of solvent in the hexagonal (Type II) form is approximately 52%. There is a single IFN-y monomer in the asymmetric
unit of structure.
This requires that the two
polypeptide chains in the rabbit IFN-y dimer have identical conformations related by a true crystallographic
and that they are
dyad axis.
Screening of potential heavy atom derivatives of the Type II crystals on the area detector is currently underway.
Suitable derivatives
solutions of K,PtCl, and K,HgI,.
have been obtained by soaking these crystals in
We are currently in the process of determining the Type II
crystal structure by the multiple isomorphous replacement technique.
ACKNOWLEDGMENTS This research was sponsored by the National Cancer Institute, DHHS, under contract no. NOl-CO-74101
with ABL-Basic
Research Program.
The contents of this publication do not
necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. REFERENCES 1. Wheelock, E.F. (1980)
Science 149, 310-311.
2. Gray, P.W., Leung, D.W., Pennica, D., Yelverton, E., Najarian, R., Simonson, C., Derynck, R., Sherwood, P.J., Wallace, D.M., Berger, S-L., Levinson, A.D. and Goeddel, D.V. (1982) Nature 295, 503-508. 3. Le, J., Barrowclough,
B.S. and Vileek, J. (1984)
J. Immunol. Methods 69, 61-70.
4. Rinderknecht, E. and Burton, L.E. (1985) In The Biology of the Interferon System (H. Kirchner, H. Schellekens, Eds.) pp. 397-402. Elsevier, Amsterdam. 5. Shalaby, M.R., Week, P.K., Rinderknecht, (1984) Cell Immunol. 81, 380-392.
E., Harkins, R.N., Frane, J.W. and Ross, M.J.
6. Czarniecki, C.W., Fennie, C.W., Powers, D.B. and Estell, D.A. (1984)
J. Virol. 49, 490-496.
7. Palladino, M.A., Sredersky, L.P., Shepard, H.M., Pearlstein, K.T., Vilcek, J. and Scheid, M.P. (1983) In Interferon Research: Clinical Application and Regulatory Consideration (K.C. Zoon, Ed.) pp. 139-147. Elsevier, Amsterdam. 8. Svredersky, L.D., Nedwin, G.E., Goeddel, D.V. and Palladino, M.A. (1985) 134. 1604-1608.
J. Immunol.
9. Stone-Wolff, D.S., Yip, Y.K., Kelker, H.C., Le, J., Henriksen-DeStefano, D., Rubin, B.Y., J. Exp. Med. 159, 828-843. Rinderknecht, E., Aggarwal, B.B. and Vilcek, J. (1984) 639
Vol.
178, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
10. Yip, Y.K., Barrowclough, B.S., Urban, C. and Vilcek, J. (1982) Science 215, 411-413. 11. Burton, L.E. Gray, P.W., Goeddel, D.V. and Rinderknecht, E. (1985) In Biology of the Interferon System, (H. Kirchner, H. Schellekens,Eds.) pp. 403-409. Elsevier, Amsterdam. 12. Chou, P.Y. and Fasman,G.D. (1974)
Biochemistry 13, 211-222.
13. Finer-Moore, J., Bazan, J.F., Rubin, J.R. and Stroud, R.M. (1989) In Prediction of Protein Structure and the Principles of Protein Conformation (G.D. Fasman,Ed.) pp. 719-759. Plenum, New York. 14. Baldwin, E.T. (1990) Ph.D. Dissertation, University of North Carolina at Chapel Hill. 15. Matthews, B.W. (1968)
J. Mol. Biol. 33, 491-497.
640
