Calcium-dependent Dimerization of Human Soluble Calcium Activated Nucleotidase
2006; Elsevier BV; Volume: 281; Issue: 38 Linguagem: Inglês
10.1074/jbc.m604413200
ISSN1083-351X
AutoresMing-Yan Yang, Katsunori Horii, Andrew B. Herr, Terence L. Kirley,
Tópico(s)Signaling Pathways in Disease
ResumoMammals express a protein homologous to soluble nucleotidases used by blood-sucking insects to inhibit host blood clotting. These vertebrate nucleotidases may play a role in protein glycosylation. The activity of this enzyme family is strictly dependent on calcium, which induces a conformational change in the secreted, soluble human nucleotidase. The crystal structure of this human enzyme was recently solved; however, the mechanism of calcium activation and the basis for the calcium-induced changes remain unclear. In this study, using analytical ultracentrifugation and chemical cross-linking, we show that calcium or strontium induce noncovalent dimerization of the soluble human enzyme. The location and nature of the dimer interface was elucidated using a combination of site-directed mutagenesis and chemical cross-linking, coupled with crystallographic analyses. Replacement of Ile170, Ser172, and Ser226 with cysteine residues resulted in calcium-dependent, sulfhydryl-specific intermolecular cross-linking, which was not observed after cysteine introduction at other surface locations. Analysis of a super-active mutant, E130Y, revealed that this mutant dimerized more readily than the wild-type enzyme. The crystal structure of the E130Y mutant revealed that the mutated residue is found in the dimer interface. In addition, expression of the full-length nucleotidase revealed that this membrane-bound form can also dimerize and that these dimers are stabilized by spontaneous oxidative cross-linking of Cys30, located between the single transmembrane helix and the start of the soluble sequence. Thus, calcium-mediated dimerization may also represent a mechanism for regulation of the activity of this nucleotidase in the physiological setting of the endoplasmic reticulum or Golgi. Mammals express a protein homologous to soluble nucleotidases used by blood-sucking insects to inhibit host blood clotting. These vertebrate nucleotidases may play a role in protein glycosylation. The activity of this enzyme family is strictly dependent on calcium, which induces a conformational change in the secreted, soluble human nucleotidase. The crystal structure of this human enzyme was recently solved; however, the mechanism of calcium activation and the basis for the calcium-induced changes remain unclear. In this study, using analytical ultracentrifugation and chemical cross-linking, we show that calcium or strontium induce noncovalent dimerization of the soluble human enzyme. The location and nature of the dimer interface was elucidated using a combination of site-directed mutagenesis and chemical cross-linking, coupled with crystallographic analyses. Replacement of Ile170, Ser172, and Ser226 with cysteine residues resulted in calcium-dependent, sulfhydryl-specific intermolecular cross-linking, which was not observed after cysteine introduction at other surface locations. Analysis of a super-active mutant, E130Y, revealed that this mutant dimerized more readily than the wild-type enzyme. The crystal structure of the E130Y mutant revealed that the mutated residue is found in the dimer interface. In addition, expression of the full-length nucleotidase revealed that this membrane-bound form can also dimerize and that these dimers are stabilized by spontaneous oxidative cross-linking of Cys30, located between the single transmembrane helix and the start of the soluble sequence. Thus, calcium-mediated dimerization may also represent a mechanism for regulation of the activity of this nucleotidase in the physiological setting of the endoplasmic reticulum or Golgi. It was recently discovered that mammals, including rats (1Failer B.U. Braun N. Zimmermann H. J. Biol. Chem. 2002; 277: 36978-36986Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) and humans (2Smith T.M. Hicks-Berger C.A. Kim S. Kirley T.L. Arch. Biochem. Biophys. 2002; 406: 105-115Crossref PubMed Scopus (60) Google Scholar), express proteins homologous to the nucleotidases used by blood-sucking insects, such as bed bugs (3Valenzuela J.G. Charlab R. Galperin M.Y. Ribeiro J.M. J. Biol. Chem. 1998; 273: 30583-30590Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), mosquitoes (4Champagne D.E. Smartt C.T. Ribeiro J.M. James A.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 694-698Crossref PubMed Scopus (219) Google Scholar), and ticks (5Mans B.J. Gaspar A.R. Louw A.I. Neitz A.W. Exp. Appl. Acarol. 1998; 22: 353-366Crossref PubMed Scopus (55) Google Scholar). The mammalian enzymes exist as both membrane-bound forms in the endoplasmic reticulum and Golgi (1Failer B.U. Braun N. Zimmermann H. J. Biol. Chem. 2002; 277: 36978-36986Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), as well as secreted, soluble forms (2Smith T.M. Hicks-Berger C.A. Kim S. Kirley T.L. Arch. Biochem. Biophys. 2002; 406: 105-115Crossref PubMed Scopus (60) Google Scholar). The physiological functions of these vertebrate nucleotidases are not yet well established, although they may play a role in protein glycosylation (1Failer B.U. Braun N. Zimmermann H. J. Biol. Chem. 2002; 277: 36978-36986Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). In insect saliva, these soluble proteins are part of the anti-coagulation mixture injected at the site of skin puncture to keep the blood of the host from clotting, so that the insect can continue to feed. However, unlike the insect members of this family, the mammalian enzymes hydrolyze ADP very poorly (1Failer B.U. Braun N. Zimmermann H. J. Biol. Chem. 2002; 277: 36978-36986Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 2Smith T.M. Hicks-Berger C.A. Kim S. Kirley T.L. Arch. Biochem. Biophys. 2002; 406: 105-115Crossref PubMed Scopus (60) Google Scholar, 6Murphy D.M. Ivanenkov V.V. Kirley T.L. Biochemistry. 2003; 42: 2412-2421Crossref PubMed Scopus (27) Google Scholar), making control of platelet activation and subsequent blood coagulation an unlikely function for the mammalian enzymes, because ADP is a critical agonist for platelet activation. However, Dai et al. (7Dai J. Liu J. Deng Y. Smith T.M. Lu M. Cell. 2004; 116: 649-659Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) were able to engineer the soluble human enzyme to hydrolyze ADP efficiently, by combining five point mutations based on their analysis of the crystal structure. Because of its substrate preference and subcellular localization, Failer et al. (1Failer B.U. Braun N. Zimmermann H. J. Biol. Chem. 2002; 277: 36978-36986Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) postulated that the membrane-bound rat enzyme may function to support glycosylation reactions related to protein biosynthetic quality control in the endoplasmic reticulum.These mammalian enzymes are termed calcium activated nucleotidases (CAN), 3The abbreviations used are: CAN, calcium activated nucleotidase (Gen-Bank™ accession number AF328554); SCAN, soluble calcium activated nucleotidase; DTT, dithiothreitol; DSS, disuccinimidyl suberate; BMH, bis-maleimidohexane; BMOE, bis-maleimidoethane; MOPS, 3-[N-morpholino] propanesulfonic acid.3The abbreviations used are: CAN, calcium activated nucleotidase (Gen-Bank™ accession number AF328554); SCAN, soluble calcium activated nucleotidase; DTT, dithiothreitol; DSS, disuccinimidyl suberate; BMH, bis-maleimidohexane; BMOE, bis-maleimidoethane; MOPS, 3-[N-morpholino] propanesulfonic acid. as described in a recent review (8Smith T.M. Kirley T.L. Purinergic Signal. 2006; 2: 327-333Crossref PubMed Scopus (16) Google Scholar). Mammalian CAN enzymes were discovered less than 5 years ago, and there have been only a few publications characterizing these enzymes (1Failer B.U. Braun N. Zimmermann H. J. Biol. Chem. 2002; 277: 36978-36986Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 2Smith T.M. Hicks-Berger C.A. Kim S. Kirley T.L. Arch. Biochem. Biophys. 2002; 406: 105-115Crossref PubMed Scopus (60) Google Scholar, 6Murphy D.M. Ivanenkov V.V. Kirley T.L. Biochemistry. 2003; 42: 2412-2421Crossref PubMed Scopus (27) Google Scholar, 7Dai J. Liu J. Deng Y. Smith T.M. Lu M. Cell. 2004; 116: 649-659Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 9Yang M. Kirley T.L. Biochemistry. 2004; 43: 9185-9194Crossref PubMed Scopus (16) Google Scholar). However, the progress on the structure of the soluble form of this nucleotidase (SCAN) was rapid, culminating in the report of the crystal structure of the human enzyme in 2004 (7Dai J. Liu J. Deng Y. Smith T.M. Lu M. Cell. 2004; 116: 649-659Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Nonetheless, some questions remain, and alternative interpretations of the crystal structure have been presented (9Yang M. Kirley T.L. Biochemistry. 2004; 43: 9185-9194Crossref PubMed Scopus (16) Google Scholar), based on the lack of calcium ions during crystallization (7Dai J. Liu J. Deng Y. Smith T.M. Lu M. Cell. 2004; 116: 649-659Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), because it was previously demonstrated that human SCAN is calcium-activated and undergoes a calcium-induced conformational change (6Murphy D.M. Ivanenkov V.V. Kirley T.L. Biochemistry. 2003; 42: 2412-2421Crossref PubMed Scopus (27) Google Scholar).There have been no published reports of CAN dimerization. In fact, the size of human SCAN, determined by size exclusion chromatography, was reported to be consistent with a monomer in two previously published works from this laboratory (2Smith T.M. Hicks-Berger C.A. Kim S. Kirley T.L. Arch. Biochem. Biophys. 2002; 406: 105-115Crossref PubMed Scopus (60) Google Scholar, 6Murphy D.M. Ivanenkov V.V. Kirley T.L. Biochemistry. 2003; 42: 2412-2421Crossref PubMed Scopus (27) Google Scholar). In addition, sedimentation equilibrium experiments conducted with 2 mm CaCl2 in a buffer that also contained 150 mm NaCl indicated that SCAN was monomeric under these conditions (7Dai J. Liu J. Deng Y. Smith T.M. Lu M. Cell. 2004; 116: 649-659Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Thus, it was unexpected when the first data suggesting that this protein forms a dimer in solution were obtained.In this study, we characterized the dimerization of human SCAN by size exclusion chromatography, analytical ultracentrifugation, chemical cross-linking, site-directed mutagenesis, and x-ray crystallography. It was observed that under appropriate solution conditions, this nucleotidase forms dimers in the presence of calcium and strontium, but not in the presence of magnesium ions. Dimers are also observed in the membrane-bound, full-length form of human CAN expressed in mammalian COS cells. In COS cells, these dimers are partially disulfide-linked in the wild-type enzyme, mediated by a cysteine close to the N terminus that is not present in the soluble form of the enzyme.The reason for the observed increased nucleotidase activities of a previously published mutation of SCAN (E130Y) (9Yang M. Kirley T.L. Biochemistry. 2004; 43: 9185-9194Crossref PubMed Scopus (16) Google Scholar) was not clear, given that this residue is far removed from the active site as identified by the crystal structure of the human enzyme co-crystallized with a nonhydrolyzable nucleotide analogue (7Dai J. Liu J. Deng Y. Smith T.M. Lu M. Cell. 2004; 116: 649-659Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). However, a likely mechanism is suggested by the location of this residue in the dimer interface identified in the current study by cysteine replacement mutations combined with sulfhydryl-specific cross-linking, as well as crystallographic analyses.The identified dimer interface has a more extensive buried surface area and better shape complementarity than other protein-protein interfaces observed in the crystal structure. Human SCAN has a five-bladed β-propeller fold (7Dai J. Liu J. Deng Y. Smith T.M. Lu M. Cell. 2004; 116: 649-659Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Our results indicate that the interface of the dimer is localized primarily within the 2d and 3d elements of the β-propeller and that the dimer interface is dominated by hydrophobic interactions.EXPERIMENTAL PROCEDURESMaterials—The QuikChange site-directed mutagenesis kit and Escherichia coli competent bacteria were purchased from Stratagene. The DNA Core Facility at the University of Cincinnati produced the synthetic oligonucleotides and sequenced all cDNA constructs. Plasmid purification kits and nickel-nitrilotriacetic acid-agars were purchased from Qiagen. The bacterial expression vector pET28a and the bacterial expression BL21(DE3) cells were purchased from Novagen. Glycerol and dialysis tubing were from Fisher. The chemical cross-linkers DSS, glutaraldehyde, BMH, and BMOE were purchased from Pierce, as were B-PER bacterial extraction reagent and the enhanced chemiluminescent reagents used for Western blots. The pre-cast SDS-PAGE 4-15% gradient mini-gels were obtained from Bio-Rad. Ampicillin, kanamycin, nucleotides, isopropyl-β-d-thiogalactopyranoside, glucose, DTT, and other reagents were from Sigma. Sephacryl S-100 used for size exclusion chromatography was obtained from Amersham Biosciences.Site-directed Mutagenesis—A series of mutants of human SCAN have been designed and made to introduce cysteine residues at various surface locations, thus allowing analysis of the ability of these mutant proteins to be intermolecularly cross-linked into dimers. There is only one native cysteine (Cys257) in the sequence of human SCAN, and to simplify the interpretation of experiments, this residue (Cys257) was mutated to serine (C257S), to serve as the starting point for the cysteine substitution mutants. This "cysteine-less" C257S mutant has wild type-like activity and characteristics. Thus, most of the following mutations were made in this C257S background using the primers listed, with the substitution sites in bold type and underlined: C257S, 5′-CCATGAGTCTGCC-AGCTGGAGTGACACGC-3′; S109C, 5′-CTGACCCTGTCAGACTGTGGGGACAAGGTG-3′; I170C, 5′-CAAAGCCGTGCCCTGGGTGTGTCTGTCCGACGGCGACGG-3′; S172C, 5′-CTGGGTGATTCTGTGCGACGGCGACGGCAC-3′; S226C, 5′-GTGGTGGGCTACAAGGGCTGTGTGGACCACGAGAACTGG-3′; S278C, 5′-CCAGGAGCGCTACTGCGAGAAGGACGACG-3′; A287C, 5′-GAGCGCAAGGGCTGCAACCTGCTGCTGAG-3′; and E130Y, 5′-GTCCCACCTGGCGTATAAGGGGAGAGGCATG-3′ (mutated in wild-type background). The complementary antisense oligonucleotides also necessary for the mutagenesis are not shown. The mutagenesis methodology was described previously (10Smith T.M. Kirley T.L. Biochim. Biophys. Acta. 1998; 1386: 65-78Crossref PubMed Scopus (164) Google Scholar) and used the QuikChange site-directed mutagenesis kit as described (11Smith T.M. Lewis Carl S.A. Kirley T.L. Biochemistry. 1999; 38: 5849-5857Crossref PubMed Scopus (56) Google Scholar). The presence of the correct mutation and lack of unwanted mutations were confirmed by automated DNA sequencing.Expression, Refolding, and Purification of SCAN Mutants in E. coli BL21(DE3) Bacterial Cells—The wild-type and mutant SCAN cDNA constructs were used to transform bacterial expression host BL21(DE3) cells (6Murphy D.M. Ivanenkov V.V. Kirley T.L. Biochemistry. 2003; 42: 2412-2421Crossref PubMed Scopus (27) Google Scholar), and after induction of expression with isopropyl-β-d-thiogalactopyranoside, bacterial inclusion bodies containing the SCAN proteins were isolated, and the protein was refolded and purified as described previously (6Murphy D.M. Ivanenkov V.V. Kirley T.L. Biochemistry. 2003; 42: 2412-2421Crossref PubMed Scopus (27) Google Scholar, 12Ivanenkov V.V. Murphy-Piedmonte D.M. Kirley T.L. Biochemistry. 2003; 42: 11726-11735Crossref PubMed Scopus (45) Google Scholar). The only modification was that DTT was added during some stages of the purification of the refolded cysteine mutant proteins to avoid cysteine oxidation. Thus, after eluting from nickel-nitrilotriacetic acid beads that bind the N-terminal His6 tag, the proteins were processed in buffer which includes 0.2 mm DTT. Even so, mutations that tend to efficiently oxidize spontaneously, such as S226C, still formed some intermolecular dimers during purification. DTT was also added to a final concentration of 0.2-0.3 mm after protein elution from the anion exchange cartridge. Prior to cross-linking experiments, residual DTT was eliminated or minimized by dialysis or by dilution and subsequent protein concentration using Amicon 30-kDa molecular mass cut-off concentrators. Protein concentrations were determined using the Bio-Rad G-250 dye binding technique according to the modifications of Stoscheck (13Stoscheck C.M. Anal. Biochem. 1990; 184: 111-116Crossref PubMed Scopus (136) Google Scholar) or using calculated molar extinction coefficients for the 37,764-Da wild-type SCAN protein of 81,360 cm-1 m-1 at 280 nm and 82,850 cm-1 m-1 for the E130Y mutant, estimated by the ProtParam tool on the ExPASy server (us.expasy.org/tools/protparam.html).Nucleotidase Assays—Nucleotidase activities were determined by measuring the amount of inorganic phosphate (Pi) released from nucleotide substrates in the presence of 5 mm Ca2+ at 37 °C using a modification of the technique of Fiske and Subbarow (14Fiske C.H. Subbarow Y. J. Biol. Chem. 1925; 66: 375-400Abstract Full Text PDF Google Scholar), as described previously (15Smith T.M. Kirley T.L. Biochemistry. 1999; 38: 321-328Crossref PubMed Scopus (100) Google Scholar). The enzyme assays were initiated by the addition of a final concentration of 2.5 mm nucleotide, except for the experiments measuring the dependence of activity on SCAN protein concentration (see Fig. 7), where a final concentration of 4 mm ADP was used. The units used for enzyme activity are μmol of Pi generated per mg of protein/h. Prior to assay, CAN protein was diluted into 50 mm Tris-HCl, pH 6.8, containing 0.1% Tween 20 to avoid adsorption to surfaces previously noted to occur when handling very dilute solutions of SCAN.Size Exclusion Chromatography—Human wild-type SCAN protein was analyzed on a 28-cm-long, 11.5-ml bed volume Sephacryl S-100 column equilibrated in 20 mm MOPS, pH 7.4, with or without 2 mm CaCl2, at a flow rate of 0.28 ml/min. 200-μl aliquots of various concentrations of SCAN protein in the column buffer were applied to the column, and the elution positions of the 280-nm peaks were noted.Chemical Cross-linking with Glutaraldehyde or DSS, a Hydrophobic, Lysine-specific Cross-linker—Cross-linking stock solutions were always freshly prepared (in dry Me2SO for DSS, in water for glutaraldehyde). Purified SCAN proteins (0.1 mg/ml) were incubated in 20 mm MOPS (pH 7.4) in the presence or absence of 5 mm CaCl2, for 10 min at 22 °C in the presence or absence of DSS (250 μm) or glutaraldehyde (500 μm). Cross-linking was stopped by adding an excess (10 mm) of lysine and incubating for 5 min at room temperature. Finally, nonreducing sample buffer was added, and the samples were boiled for 5 min, run on SDS-PAGE, and analyzed either by Coomassie staining of the gel or by Western blot. When analyzed by Western blot, an affinity-purified anti-CAN anti-peptide antibody raised against the C-terminal sequence was used, and the bands were detected by enhanced chemiluminescence, as described previously (2Smith T.M. Hicks-Berger C.A. Kim S. Kirley T.L. Arch. Biochem. Biophys. 2002; 406: 105-115Crossref PubMed Scopus (60) Google Scholar).Analysis of Cysteine Substitution Mutants by Cross-linking Using the Hydrophobic, Sulfhydryl-specific Reagents BMH and BMOE—Stocks of 20 mm BMH and BMOE were made in dry Me2SO and stored at-80 °C. The final concentrations of these reagents used for cross-linking were varied from 6 to 50 μm. The cross-linking reactions were carried out in 20 mm MOPS, pH 7.4, at 22 °C, at a final protein concentration of 0.1 mg/ml, both in the presence and absence of 5 mm CaCl2. BMH or BMOE (or Me2SO used as a vehicle control) was added and incubated for 10 min at 22 °C. The cross-linking reactions were stopped by adding an excess of cysteine and incubating for 5 min prior to adding nonreducing SDS-PAGE sample buffer. After heating the samples for 10 min at 60 °C, SDS-PAGE was performed as described above.Calcium-induced Change in the Tryptophan Fluorescence of SCAN as a Function of Protein Concentration—The increase in tryptophan fluorescence (excitation at 295 nm, emission at 340 nm) was measured after a single addition of CaCl2 to a final Ca2+ concentration of 2 mm in 20 mm MOPS buffer, pH 7.4 (6Murphy D.M. Ivanenkov V.V. Kirley T.L. Biochemistry. 2003; 42: 2412-2421Crossref PubMed Scopus (27) Google Scholar). The protein concentration was varied from a low near the sensitivity limit of the instrument, to a high at approximately the concentration where inner filter effects and other nonideal fluorescence effects are observed. To allow comparison with previous studies and to the current analytical ultracentrifugal studies, the experiments were performed in the presence and absence of 50 mm NaCl.Analytical Ultracentrifugation Analysis—Sedimentation velocity and equilibrium experiments were performed at 20 °C in a Beckman XL-I ProteomeLab analytical ultracentrifuge equipped with an absorbance optical system as described before (16Herr A.B. White C.L. Milburn C. Wu C. Bjorkman P.J. J. Mol. Biol. 2003; 327: 645-657Crossref PubMed Scopus (76) Google Scholar). For these experiments, two different buffers were used; Buffer A (20 mm MOPS, pH 7.4) is identical to that used previously to monitor absorbance and fluorescence changes induced by Ca2+ (6Murphy D.M. Ivanenkov V.V. Kirley T.L. Biochemistry. 2003; 42: 2412-2421Crossref PubMed Scopus (27) Google Scholar) and very similar to that used for nucleotidase activity assays, whereas Buffer B (20 mm MOPS, pH 7.4, 50 mm NaCl) is designed to minimize thermodynamic nonideality caused by charge-charge interactions during sedimentation (Table 1). For sedimentation velocity experiments, 0.85 μm wild-type SCAN was analyzed in the absence and the presence of a divalent cation (2 or 20 mm MgCl2,2 mm CaCl2 or SrCl2) or in the presence of monovalent cation (150 or 300 mm NaCl) at 48,000 rpm. Sedimentation coefficient distributions were determined using the program Sedfit (17Schuck P. Biophys. J. 2000; 78: 1606-1619Abstract Full Text Full Text PDF PubMed Scopus (3004) Google Scholar). For sedimentation equilibrium experiments, the wild-type and E130Y mutant SCANs were analyzed in the absence and the presence of 0.2 mm or2mm CaCl2. Samples at three concentrations (0.25, 1.25, and 2.50 μm) in Buffer A were centrifuged at three different speeds (15,000, 18,000, and 26,000 rpm), whereas samples in Buffer B at two protein concentrations (1.25 and 2.50 μm) were centrifuged at five different speeds (15,000, 18,000, 21,000, 25,000, and 36,000 rpm). For each calcium concentration, a global analysis of the resultant nine or ten data sets was performed using the program WinNONLIN (Jeff Lary, University of Connecticut, Storrs, CT).TABLE 1Apparent SCAN dimerization dissociation constants, KD (all values in μm), derived from equilibrium sedimentation experiments in the presence and absence of NaCl and divalent cationsBufferDimerization constant (95% CI) in μmWild-type SCANE130Y SCAN20 mm MOPS 7.4 + 0.2 mm CaCl20.992 (0.670-1.44)20 mm MOPS 7.4 + 2 mm CaCl20.170 (0.109-0.259)20 mm MOPS 7.4 + 20 mm CaCl20.142 (0.086-0.228)20 mm MOPS 7.4 + 2 mm SrCl20.0306 (0.0146-0.0595)20 mm MOPS 7.4/50 mm NaCl + 0 mm CaCl21160 (333-9060)11.8 (9.34-14.9)20 mm MOPS 7.4/50 mm NaCl + 0.2 mm CaCl241.9 (31.4-56.5)1.69 (1.33-2.13)20 mm MOPS 7.4/50 mm NaCl + 2 mm CaCl20.563 (0.384-0.799)0.179 (0.107-0.283) Open table in a new tab Full-length CAN Expression in Mammalian COS Cells—COS-1 cells were transfected with 4 μg of plasmid DNA encoding wild-type or mutant CAN proteins/100-mm plate using Lipofectamine and PLUS reagents as described previously (10Smith T.M. Kirley T.L. Biochim. Biophys. Acta. 1998; 1386: 65-78Crossref PubMed Scopus (164) Google Scholar). Transfection with an empty pcDNA3 vector was also performed as a control. Approximately 48 h post-transfection, the COS-1 cells were harvested, and the crude cell membrane preparations were obtained as described (10Smith T.M. Kirley T.L. Biochim. Biophys. Acta. 1998; 1386: 65-78Crossref PubMed Scopus (164) Google Scholar). In some experiments, the alkylating agent, N-ethylmaleimide, was added to the buffer used to homogenize the crude COS cell membranes (final concentration, 2 mm) to prevent oxidation of free sulfhydryls to disulfides during membrane preparation.Crystallization and Structure Determination of Wild-type and E130Y Human SCAN—Crystallization was performed using a sitting drop vapor diffusion method at 293 K in 96-well plates. The crystallization conditions for both wild-type and E130Y SCAN were very similar to that of the wild-type protein crystallized with strontium (7Dai J. Liu J. Deng Y. Smith T.M. Lu M. Cell. 2004; 116: 649-659Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). For wild-type SCAN, 1 μl of protein solution (5.9 mg/ml in 20 mm MOPS, pH 7.4, and 0.1 m NaCl) was mixed with 1 μl of reservoir solution (8% polyethylene glycol 4000, 0.2 m ammonium sulfate, 0.1 m sodium acetate, pH 4.8, and 10 mm CaCl2) and equilibrated against 100 μl of reservoir solution. For the E130Y mutant,1 μl of protein solution (4.9 mg/ml in 20 mm MOPS, pH 7.4 and 0.1 m NaCl) was mixed with 1 μl of reservoir solution (16% polyethylene glycol 4000, 0.2 m ammonium sulfate, 0.1 m sodium acetate, pH 4.8, and 10 mm CaCl2) and equilibrated against 100 μl reservoir solution. The crystals were cryoprotected by transfer to a mother-liquor solution containing 15% glycerol and flash frozen in a cold nitrogen stream maintained at 110 K. For the wild-type crystal, the data were collected in-house using a Rigaku Micromax-007 generator and an R-Axis IV++ image plate detector. 1080 images were collected as 0.5° oscillations with 10 min of exposure/frame, using two chi orientations (0° and 30°) to give a total of 540° of data with high redundancy. The data, which extended to 2.3 Å resolution, were processed with Crystal-Clear and belonged to space group P1, with the unit-cell parameters a = 43.09 Å, b = 52.37 Å, c = 77.43 Å, α = 75.06°, β = 74.52°, γ = 79.47°. The data for the E130Y crystal were collected at Beamline SER-22ID at the Advanced Photon Source, Argonne National Laboratory. A total of 720 images were collected as 0.5° oscillations at λ = 0.97182 Å and processed with the HKL2000 suite. The crystal diffracted to 2.4 Å resolution and belonged to space group P1, with the unit-cell parameters a = 42.89 Å, b = 52.41 Å, c = 77.81 Å, α = 100.94°, β = 106.51°, γ = 99.32°. The crystallographic parameters and statistics of the data collections are listed in Table 3. For both crystals, initial phasing was obtained with Phaser1.3.1 using the published coordinates of wild-type SCAN crystallized with strontium as a search model (Protein Data Bank code 1S1D). In both crystals, two molecules were found in the asymmetric unit, which are related by 2-fold noncrystallographic symmetry. All of the crystallographic refinements were performed with Refmac5 (18Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13774) Google Scholar). Fig. 9 was created using Pymol. Analyses of buried surface area and surface complementarity were carried out using Areaimol and Sc, respectively, from the CCP4 suite. The data collection and refinement statistics are reported in Table 3.TABLE 3SCAN crystallographic data collection and refinement statisticsData collectionWild type (Protein Data Bank 2H2N)E130Y (Protein Data Bank 2H2U)SourceRigaku RA-Micro007APS SER-22IDWavelength (Å)1.541780.97182Space groupP1P1Unit cell a, b, c (Å)43.09, 52.37, 77.4342.89, 52.41, 77.81α, β, γ (°)75.06, 74.52, 79.47100.94, 106.51, 99.32Resolution range (Å)aThe values in parentheses refer to the highest resolution shell.31.08-2.30 (2.38-2.30)50.00-2.40 (2.49-2.40)Completeness (%)99.7 (99.3)86.8 (83.8)Reflections (observed/unique)150511/2772736128/24181Average redundancy5.43 (5.13)1.70 (1.50)Average I/σ(I)17.9 (4.9)13.2 (2.81)Rmerge (%)bRmerge = ∑|I - 〈I 〉|/∑I.5.8 (17.8)6.5 (45.1)Model refinementResolution range (Å)31.08-2.3050.00-2.40Reflections (work/test)26332/139519868/1092Rwork/Rfree (%)c∑||Fo| - |Fc||/∑|Fo| for all data except for 5%, which was used for the Rfree calculation.16.8/24.921.3/29.9Root mean square deviations from idealityBond lengths (Å)0.0270.012Bond angles (°)2.1361.488No. of residues in molecule (A/B)317/315315/317Missing residues in molecule (A/B)22/2424/22No. of water molecules22835No. of calcium ions22No. of acetate ions10Ramachandran plotResiduesMost favored486 residues, 89.0%466 residues, 85.3%Additional allowed60 residues, 11.0%76 residues, 13.9%Generously allowed0 residues, 0%4 residues, 0.7%a The values in parentheses refer to the highest resolution shell.b Rmerge = ∑|I - 〈I 〉|/∑I.c ∑||Fo| - |Fc||/∑|Fo| for all data except for 5%, which was used for the Rfree calculation. Open table in a new tab FIGURE 9Crystal structures of wild-type and E130Y human SCAN dimers. A, crystal structure of the dimer of human SCAN crystallized in ammonium sulfate (see "Experimental Procedures"). The dimer interface is located along a 2-fold noncrystallographic symmetry axis such that residues primarily from the 1d′, 2d, and 3d strands from each protomer interact across the interface. B, chemical nature of the dimer interface in the E130Y mutant, shown looking through molecule A at the surface of molecule B. The side chains and dimer interface surface are colored according to the chemical nature of the residues that interact in the dimer: yellow for hydrophobic, green for polar, blu
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