THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 262, No. 24, Issue of August 25. pp. 118&1-11885,19S7 Printed in USA. Crystallization and Preliminary X-ray Investigation of Sarcoplasmic Calcium-binding Protein from Nereis diuersicoZor* (Received for publication, April 7, 1987) Y. Sudhakar BabuSs, Jos A. Coxll, and William J. Cook@11 From the 1) Department of Pathology, $Center for Macromolecular Crystallography, and $Comprehensive Cancer Center, University of Alabama at Birmingham. University Station, Birmingham, Alabama 35294 and the VDepartment of Biochemistry, Universi& of Geneva, 121 I Geneva 4, Switzerla& Crystals of sarcoplasmic calcium-binding proteins from Nereis diversicolor have been grown from solu- tions of ammonium sulfate. The crystals are mono- clinic, space group P21; the axes are a = 43.65 (l), b = 56.05 (l), c = 65.77 (1) A, and B = 92.58 (2)”. The crystals are quite stable to x-rays and diffract beyond 2.5 A resolution. The asymmetric unit contains two protein molecules. Sarcoplasmic calcium-binding proteins (SCPs)’ are an im- portant Ca2+-binding system in both vertebrate and inverte- brate muscle (1). Those found in vertebrates are called parv- albumins and belong to the family of small, acidic, intracel- lular Ca2+-binding proteins that includes calmodulin and tro- ponin C. The SCPs found in invertebrates display properties distinct from those of parvalbumins, although they share some sequence homology. Unlike parvalbumins, which are essen- tially confined to striated skeletal muscle, SCPs are present in a wide range of muscle types. The function of SCPs is not yet known. SCPs from two species of sandworm have been isolated and sequenced: Nereis diuersicolor (2,3) and Perinereis uancaurica tetradentutu (4). Each is a single polypeptide chainthat con- tains 174 amino acids and has a molecular weight of about 19,500. There are 23 amino acid differences between the two proteins. It has been shown that sandworm SCP has three CaZ+-Mg2+ mixed sites, although Ca2+ uptake causes a release of Mg2+ (2). Sequence homology of sandworm SCPs with Ca2+-binding proteins of known three-dimensional structure suggests that they originally contained four Ca2+-binding do- mains with the so-called EF-hand structure (5). Three EF- hand type Ca2+-binding domains (I, 111, and IV) can be iden- tified in each protein by sequence homology with related Ca2+- binding proteins. The portionof the sequence corresponding to domain I1 has had a number of amino acid replacements and deletions and probably does not bind Ca2+. EXPERIMENTAL PROCEDURES Nereis SCP was isolated and purified as described by Cox and Stein (2). For crystallization experiments, the lyophilized material was dissolved in distilled water containing 2.5 mM CaC12. Crystals were obtained by vapor diffusion equilibration of 2-pl drops hanging *This research was supported by National Institutes of Health Grants GM-29427 and CA-13148, Swiss National Science Foundation Grant 3.161.0.85, and the Muscular Dystrophy Association of Amer- ica. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ’ The abbreviations used are: SCPs, sarcoplasmic calcium-binding proteins; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. from siliconized coverslips inverted on Linbro plates. The drops consisted of 1 pl of a solution containing 15 mg of SCP/ml of distilled water and 2.5 mM CaC12, plus 1 pl of a solution containing 72% ammonium sulfate in 0.05 M HEPES buffer, pH 7.6. These drops were equilibrated against 1 mlof a solution containing 72% ammo- nium sulfate in 0.05 M HEPES buffer, pH 7.6. After 6-8 days at 22 “C, small monoclinic plates with dimensions up to 0.06 X 0.06 X 0.02 mm were obtained. However, these never grewlarger than about 0.1 mm on edge, so seeding was used. A single, medium-size crystal was added to a 24 drop containing 15 mg of SCP/ml, 2.5 mM CaC12, and 63% ammonium sulfate in 0.05 M HEPES buffer, pH 7.8. These drops were equilibrated against 1 ml of a solution containing 63% ammonium sulfate in 0.05 M HEPES buffer, pH 7.8. After 2-3 days at 22 “C, large, monoclinic prisms with dimensions up to 0.4 X 0.4 X 0.3 mm were obtained. For x-ray studies, crystals are transferred to a stabilizing solution of 80% ammonium sulfate in 0.05 M HEPES buffer, pH 7.8. RESULTS AND DISCUSSION X-ray precession photographs show that the crystals are monoclinic; space group P2, is indicated by the systematic absence of reflections OkO with k odd (Fig. 1). Unit cell parameters, which were obtained by least-squares refinement of the angular settings for 25 reflections measured on a diffractometer, are a = 43.65 (l), b = 56.05 (l), c = 65.77 (1) A, and /3 = 92.58 (2)O. Assuming an occ‘lpancy of two 19,500- dalton chains per crystallographic asymmetric unit, and a .. FIG. 1. Precession photograph of the hk0 zone of Nereis SCP, = 15’ (3.0 A resolution). 11884
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THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 262, No. 24, Issue of August 25. pp. 118&1-11885,19S7 Printed in U S A .
Crystallization and Preliminary X-ray Investigation of Sarcoplasmic Calcium-binding Protein from Nereis diuersicoZor*
(Received for publication, April 7, 1987)
Y. Sudhakar BabuSs, Jos A. Coxll, and William J. Cook@11 From the 1) Department of Pathology, $Center for Macromolecular Crystallography, and $Comprehensive Cancer Center, University of Alabama at Birmingham. University Station, Birmingham, Alabama 35294 and the VDepartment of Biochemistry, Universi& of Geneva, 121 I Geneva 4, Switzerla&
Crys ta l s of sarcoplasmic calcium-binding proteins f rom Nereis diversicolor have been grown f rom solu- tions of ammonium sulfate. The crystals are mono- clinic, space group P21; the axes are a = 43.65 (l), b = 56.05 (l), c = 65.77 (1) A, and B = 92.58 (2)”. The crystals are quite stable to x-rays and di f f rac t beyond 2.5 A resolution. The a symmet r i c unit contains two protein molecules.
Sarcoplasmic calcium-binding proteins (SCPs)’ are an im- portant Ca2+-binding system in both vertebrate and inverte- brate muscle (1). Those found in vertebrates are called parv- albumins and belong to the family of small, acidic, intracel- lular Ca2+-binding proteins that includes calmodulin and tro- ponin C . The SCPs found in invertebrates display properties distinct from those of parvalbumins, although they share some sequence homology. Unlike parvalbumins, which are essen- tially confined to striated skeletal muscle, SCPs are present in a wide range of muscle types. The function of SCPs is not yet known.
SCPs from two species of sandworm have been isolated and sequenced: Nereis diuersicolor (2,3) and Perinereis uancaurica tetradentutu (4). Each is a single polypeptide chain that con- tains 174 amino acids and has a molecular weight of about 19,500. There are 23 amino acid differences between the two proteins. I t has been shown that sandworm SCP has three CaZ+-Mg2+ mixed sites, although Ca2+ uptake causes a release of Mg2+ (2). Sequence homology of sandworm SCPs with Ca2+-binding proteins of known three-dimensional structure suggests that they originally contained four Ca2+-binding do- mains with the so-called EF-hand structure (5) . Three EF- hand type Ca2+-binding domains (I, 111, and IV) can be iden- tified in each protein by sequence homology with related Ca2+- binding proteins. The portion of the sequence corresponding to domain I1 has had a number of amino acid replacements and deletions and probably does not bind Ca2+.
EXPERIMENTAL PROCEDURES
Nereis SCP was isolated and purified as described by Cox and Stein (2). For crystallization experiments, the lyophilized material was dissolved in distilled water containing 2.5 mM CaC12. Crystals were obtained by vapor diffusion equilibration of 2-pl drops hanging
*This research was supported by National Institutes of Health Grants GM-29427 and CA-13148, Swiss National Science Foundation Grant 3.161.0.85, and the Muscular Dystrophy Association of Amer- ica. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
’ The abbreviations used are: SCPs, sarcoplasmic calcium-binding proteins; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.
from siliconized coverslips inverted on Linbro plates. The drops consisted of 1 pl of a solution containing 15 mg of SCP/ml of distilled water and 2.5 mM CaC12, plus 1 pl of a solution containing 72% ammonium sulfate in 0.05 M HEPES buffer, pH 7.6. These drops were equilibrated against 1 ml of a solution containing 72% ammo- nium sulfate in 0.05 M HEPES buffer, pH 7.6. After 6-8 days at 22 “C, small monoclinic plates with dimensions up to 0.06 X 0.06 X 0.02 mm were obtained. However, these never grew larger than about 0.1 mm on edge, so seeding was used. A single, medium-size crystal was added to a 2 4 drop containing 15 mg of SCP/ml, 2.5 mM CaC12, and 63% ammonium sulfate in 0.05 M HEPES buffer, pH 7.8. These drops were equilibrated against 1 ml of a solution containing 63% ammonium sulfate in 0.05 M HEPES buffer, pH 7.8. After 2-3 days at 22 “C, large, monoclinic prisms with dimensions up to 0.4 X 0.4 X 0.3 mm were obtained. For x-ray studies, crystals are transferred to a stabilizing solution of 80% ammonium sulfate in 0.05 M HEPES buffer, pH 7.8.
RESULTS AND DISCUSSION
X-ray precession photographs show that the crystals are monoclinic; space group P2, is indicated by the systematic absence of reflections OkO with k odd (Fig. 1). Unit cell parameters, which were obtained by least-squares refinement of the angular settings for 25 reflections measured on a diffractometer, are a = 43.65 (l), b = 56.05 (l) , c = 65.77 (1) A, and /3 = 92.58 (2)O. Assuming an occ‘lpancy of two 19,500- dalton chains per crystallographic asymmetric unit, and a
. .
FIG. 1. Precession photograph of the hk0 zone of Nereis SCP, = 15’ (3.0 A resolution).
11884
Crystal Data for Sarcoplasl;
partial specific volume of 0.74 ml/g, V,,, = 2.06 A3/dalton (6), and the solvent volume fraction is 40%. For one molecule in the asymmetric unit, the corresponding values are 4.12 A 3 /
dalton and 70%, respectively, so it appears likely that the asymmetric unit contains two protein molecules.
The crystals are stable indefinitely in the artificial mother liquor at room temperature. They diffract to at least 2.5 A resolution on a rotating anode and show very little decompo- sition in the x-ray beam after 4 days of data collection on a diffractometer. Therefore, they are suitable for high resolution x-ray analysis.
nic Cu2+-binding Protein 11885
REFERENCES 1. Wnuk, W., Cox, J. A., and Stein, E. A. (1982) in Calcium and
Cell Function (Cheung, W. Y., ed) vol. 2, pp. 243-278, Academic Press, Orlando, FL
2. Cox, J. A., and Stein, E. A. (1981) Biochemistry 20, 5430-5436 3. Theibert, J. L., Collins, J. H., and Cox, J. A. (1986) Fed. Proc.
4. Kobayashi, T., Takasaki, Y., Takagi, T., and Konishi, K. (1984)
5. Kretsinger, R. H., and Nockolds, C. E. (1973) J. Biol. Chem. 248,
6. Matthews, B. W. (1968) J. Mol. Biol. 33, 491-497
45, 1695 (abstr. 1255)
Eur. J. Biochem. 144, 401-408
3313-3326
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