Structure of Human Spindlin1

Spindlin1, a meiotic spindle-binding protein that is highly expressed in ovarian cancer cells, was first identified as a gene involved in gametogenesis. It appeared to be a target for cell cycle-dependent phosphorylation and was demonstrated to disturb the cell cycle. Here we report the crystal structure of human spindlin1 to 2.2Å of resolution, representing the first three-dimensional structure from the spin/ssty (Y-linked spermiogenesis-specific transcript) gene family. The refined structure, containing three repeats of five/four anti-parallel β-strands, exhibits a novel arrangement of tandem Tudor-like domains. Two phosphate ions, chelated by Thr-95 and other residues, appear to stabilize the long loop between domains I and II, which might mediate the cell cycle regulation activity of spindlin1. Flow cytometry experiments indicate that cells expressing spindlin1 display a different cell cycle distribution in mitosis, whereas those expressing a T95A mutant, which had a great decrease in phosphorous content, have little effect on the cell cycle. We further identified associations of spindlin1 with nucleic acid to provide a biochemical basis for its cell cycle regulation and other functions. Spindlin1, a meiotic spindle-binding protein that is highly expressed in ovarian cancer cells, was first identified as a gene involved in gametogenesis. It appeared to be a target for cell cycle-dependent phosphorylation and was demonstrated to disturb the cell cycle. Here we report the crystal structure of human spindlin1 to 2.2Å of resolution, representing the first three-dimensional structure from the spin/ssty (Y-linked spermiogenesis-specific transcript) gene family. The refined structure, containing three repeats of five/four anti-parallel β-strands, exhibits a novel arrangement of tandem Tudor-like domains. Two phosphate ions, chelated by Thr-95 and other residues, appear to stabilize the long loop between domains I and II, which might mediate the cell cycle regulation activity of spindlin1. Flow cytometry experiments indicate that cells expressing spindlin1 display a different cell cycle distribution in mitosis, whereas those expressing a T95A mutant, which had a great decrease in phosphorous content, have little effect on the cell cycle. We further identified associations of spindlin1 with nucleic acid to provide a biochemical basis for its cell cycle regulation and other functions. Spindlin, a major maternal transcript in Mus musculus, was named for its association and co-migration with the meiotic spindle in the first meiotic cell cycle (1Oh B. Hwang S.Y. Solter D. Knowles B.B. Development. 1997; 124: 493-503PubMed Google Scholar). It can be periodically phosphorylated during meiosis, which modulates its ability to associate with the meiotic spindle (2Oh B. Hampl A. Eppig J.J. Solter D. Knowles B.B. Mol. Reprod. Dev. 1998; 50: 240-249Crossref PubMed Scopus (37) Google Scholar). The modification of spindlin depends at least partially on the Mos/mitogen-activated protein kinase pathway (2Oh B. Hampl A. Eppig J.J. Solter D. Knowles B.B. Mol. Reprod. Dev. 1998; 50: 240-249Crossref PubMed Scopus (37) Google Scholar), which is controlled by meiotic checkpoint proteins cyclin B and Cdc2 (3Oita E. Harada K. Chiba K. J. Biol. Chem. 2004; 279: 18633-18640Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 4Frank-Vaillant M. Haccard O. Ozon R. Jessus C. Dev. Biol. 2001; 231: 279-288Crossref PubMed Scopus (23) Google Scholar). As an ovarianspecific protein, its role in sperm development seems to be fulfilled by ssty (Y-linked spermiogenesis specific transcript) (1Oh B. Hwang S.Y. Solter D. Knowles B.B. Development. 1997; 124: 493-503PubMed Google Scholar), a multicopy testis-specific spermatogenesis gene on the long arm of mouse Y chromosome whose dosage reduction was suggested to cause deformed sperm heads and infertility (5Burgoyne P.S. Mahadevaiah S.K. Sutcliffe M.J. Palmer S.J. Cell. 1992; 71: 391-398Abstract Full Text PDF PubMed Scopus (154) Google Scholar, 6Toure A. Grigoriev V. Mahadevaiah S.K. Rattigan A. Ojarikre O.A. Burgoyne P.S. Genomics. 2004; 83: 140-147Crossref PubMed Scopus (39) Google Scholar, 7Toure A. Szot M. Mahadevaiah S.K. Rattigan A. Ojarikre O.A. Burgoyne P.S. Genetics. 2004; 166: 901-912Crossref PubMed Scopus (80) Google Scholar). These two genes share more than 50% identity in amino acid sequence (Fig. 1), and together they form a new spin/ssty gene family. Homologues of spin/ssty family genes are found in Rattus norvegicus, Xenopus laevis, and Oryzias latipes but not in Drosophila melanogaster or Caenorhabditis elegans, indicating that this gene family is restricted to vertebrates (8Staub E. Mennerich D. Rosenthal A. Genome Biol. 2002; 3 (RESEARCH0003)PubMed Google Scholar). Furthermore, bioinformatics analysis suggests that the spin/ssty family proteins are composed of three repeats of a new protein motif ∼50 amino acids in length (8Staub E. Mennerich D. Rosenthal A. Genome Biol. 2002; 3 (RESEARCH0003)PubMed Google Scholar). Spindlin1, a human homolog of spindlin, has been shown to be related to ovarian cancer (9Yue W. Sun L.Y. Li C.H. Zhang L.X. Pei X.T. Ai Zheng. 2004; 23: 141-145PubMed Google Scholar). Its expression is up-regulated in ovarian cancer cells but not in normal tissues and was found to localize in cell nuclei. The transfected cells, which are prone to grow as cancer cells in nude mice, showed a complete morphological change (10Gao Y. Yue W. Zhang P. Li L. Xie X. Yuan H. Chen L. Liu D. Yan F. Pei X. Biochem. Biophys. Res. Commun. 2005; 335: 343-350Crossref PubMed Scopus (39) Google Scholar). Overexpression of spindlin1 might lead to variations in cell cycle distribution during mitosis, which is consistent with previous reports for other spindlin1 homologs (11Fletcher B.S. Dragstedt C. Notterpek L. Nolan G.P. Leukemia. 2002; 16: 1507-1518Crossref PubMed Scopus (38) Google Scholar). Although previous studies indicate that members of the spin/ssty protein family play important roles in tumorigenesis and early embryogenesis, their biochemical functions and mechanisms are largely unknown. Here we report the crystal structure of human spindlin1 at 2.2 Å of resolution, which represents the first crystal structure from the vertebrate spin/ssty gene family. The structure consists of three tandem repeats of Tudor-like domains, which represent a novel tandem repeat fold. We identify Thr-95 as important for the function of spindlin1, which is a key residue for the coordination of two phosphate ions. In addition, our preliminary biochemical data also indicate that spindlin1 associates with nucleic acid. These data provide a structural basis for further biochemical analysis of spindlin1 and the spin/ssty gene family. Protein Expression, Purification, Crystallization, and Mutagenesis—The protocols for purification and crystallization of human spindlin1 have been described previously (12Jiang F. Zhao Q. Qin L. Pang H. Pei X. Rao Z. Protein Pept. Lett. 2006; 13: 203-205Crossref PubMed Scopus (12) Google Scholar). The T95A mutant was generated using GCA to replace original codon by PCR and purified similar to the wild type protein. Both wild type spindlin1 and the T95A mutant were constructed into pCDNA3.1 myc/his(-) for cell cycle analysis. Heavy Atom Derivatization—Crystals of spindlin1 were transferred into a solution containing 35% polyethylene glycol 6000, 100 mm Tris-HCl, pH 8.0, in a stepwise manner. The mercury derivative was obtained by soaking spindlin1 crystals in this buffer supplemented with 1 mm ethyl mercuric phosphate ((C2H5HgO)HPO2) for 3 days. Data Collection and Processing—Crystals of spindlin1 belong to the space group P212121, with unit cell parameters a = 40.8 Å, b = 84.9 Å, c = 136.6 Å, α = β = γ = 90°. The crystals contain two molecules per asymmetric unit. Native data up to 2.2 Å were collected from a flash-cryocooled crystal with 25% (v/v) glycerol used as a cryoprotectant. The single-wavelength anomalous diffraction data were collected using a Rigaku RU2000 rotating CuKα anode source to 2.3 Å using a single mercury-spindlin1 crystal. Data were indexed and scaled using HKL2000 and SCALEPACK (13Otwinowski Z. Minor W. Macromolecular Crystallography, Part A. 276. Academic Press, Inc., New York1997: 307-326Google Scholar), and the unit cell dimensions were determined to be a = 40.3 Å, b = 76.2 Å, c = 136.6 Å, α = β = γ = 90°. Structure Determination and Refinement—Phases for the mercury-spindlin1 crystal were initially determined by the single-wavelength anomalous diffraction technique using CNS (14Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16919) Google Scholar). The phasing power was calculated as 2.4, and three heavy atom sites were independently located by the heavy atom search routine, yielding an overall figure of merit of 0.41 after calculation of initial single-wavelength anomalous diffraction phases at 3.0 Å. The resolution was extended to 2.3 Å using CNS. After solvent flipping, the quality of the initial electron density maps was greatly improved. Initial manual model building and fitting were carried out using 2.2 Å-resolution native data in O (15Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard Acta Crystallogr. A. 1991; 47: 110-119Crossref PubMed Scopus (12999) Google Scholar). Positional refinement, B-factor refinement, and water molecules were added using CNS. Data collection, processing, phasing, and refinement statistics are given in Table 1.TABLE 1Crystallographic data collection and refinement statisticsNativeEthyl mercuric phosphate derivativeData collectionSpace groupP212121P212121Unit cella (Å)40.840.3b (Å)84.976.2c (Å)136.6136.6Resolution (Å)50-2.2 (2.28-2.20)50-2.3 (2.38-2.30)Completeness (%)99.9 (100)97.9 (96.7)ReflectionsTotal444,671344,044Unique25,04218,996Redundancy17.8 (17.5)18.1 (17.9)RmergeaRmerge = ∑h∑i|Ii(h)- 〈I(h) 〉|∑h∑iIi(h), where 〈I 〉(h) is the mean of the observations Ii(h) of reflection h. (%)5.6 (37.4)9.0 (48.0)I/σ(I)30.8 (7.9)30.9 (7.0)Refinement statisticsResolution (Å)2.2R-factorbRwork = ∑h(||Fobs|-|Fcalc||)/∑Ih|Fobs; Rfree is the R factor for a subset (10%) of reflections that was selected before refinement calculations and not included in the refinement. (%)Working set21.4Test set26.8Root mean square deviationBonds (Å)0.012Angles (°)1.72Ramachandran plotcRamachandran plots were generated using PROCHECK. (%)Most favored86.5Allowed12.6Generously allowed0.3Disallowed0.6a Rmerge = ∑h∑i|Ii(h)- 〈I(h) 〉|∑h∑iIi(h), where 〈I 〉(h) is the mean of the observations Ii(h) of reflection h.b Rwork = ∑h(||Fobs|-|Fcalc||)/∑Ih|Fobs; Rfree is the R factor for a subset (10%) of reflections that was selected before refinement calculations and not included in the refinement.c Ramachandran plots were generated using PROCHECK. Open table in a new tab Cell Cycle Analysis—The cell cycle phase distribution of HeLa cells was examined by flow cytometry using FACScan and Cell Quest software (BD Biosciences). 1 × 106 cells were cotransfected with pBB14 (green fluorescent protein) and pCDNA3.1 myc/his(-) vector, spindlin1 wild type, or spindlin1 point mutants. After incubation in full media for 20 h, HeLa cells were harvested and prefixed using 0.5% paraformaldehyde. The cells were washed with and in for 1 h. The cells were with for after and purified of of used of and a constructed spindlin1 of were were in 20 mm pH 8.0, mm mm 1 mm and to of After 20 of incubation at 1 of buffer containing was added to the were at at a of in and with Structure of into crystal structure of spindlin1 was determined by single-wavelength anomalous diffraction from a single crystal with The initial model was into an electron density calculated to 2.3 Å resolution, and data from a native crystal model refinement to 2.2 Å. The two spindlin1 molecules in asymmetric unit were in two and in (Fig. and and in residues, electron density was for the other Spindlin1, with dimensions of × × Å, exhibits an structure that consists of and two The structure is into three structural domains that are composed of The three domains, which to and of and The are located in II, of its first and (Fig. a and As shown in II, and a similar M. Haccard O. Ozon R. Jessus C. Dev. Biol. 2001; 231: 279-288Crossref PubMed Scopus (23) Google and the of their can be with mean square of Å to and Å to is composed of two anti-parallel and from and from and from The two β of are with the first of to the and the of The long β and are by located at amino acid and and and represent the of and after their The two are located in whereas is and and they the of together with and These two are II, with in in and in in and and in with a of These two form a with between and of and and of the three domains a sequence identity of with of and (Fig. These to the of or for the the three domains. The and are important for the protein fold. and are for the of the first in and form between the first and in whereas the of and be for the of and are a of other and that are on the of the protein, but their structural roles are (Fig. II, and a New Tudor-like search for structural to the domains II, and structural homologs for different and suggested domains and repeat domains as The spindlin domains and domains two that are up of anti-parallel β The of the of domains in the two are by a that the of the (Fig. Furthermore, that in the Tudor-like are highly between domains II, and of spindlin1 and the (Fig. a search not for the overall structure, the spindlin1 structure is a novel arrangement of Tudor-like domains. Although spin/ssty repeats and domains share similar are between of domains showed in the of the The of the of the spindlin1 domains is from that of the domains. In addition, of the a mean square of Å, indicating between the two of domains. Furthermore, previous studies have shown that the loop and the loop together with the between two are of great for its but the residue in the spindlin domains is not with the domains (Fig. and of observations that spin/ssty repeats might have a different function or at least function in a different than domains. The between in Structure and the crystal structure, are two molecules in asymmetric unit that are with a mean square of Å for of the crystal the of a major between the two spindlin1 spindlin1 two and for association with is by and the loop whereas B is by and the and The two spindlin1 molecules associate an that of and B of the of which and the between and B of which to stabilize the the first of in an anti-parallel with of I in B. As a and in B in the of the of of its It be that is composed of β which is than I and II, and this structure is by from the in a similar to and in the first two the of this spin/ssty repeat is at least in by The also and in B and and in A. In addition, the two anti-parallel between the two molecules also lead to the of at least and and is which also to the (Fig. a and with our structure, spindlin1 a in as by and not using glycol as also showed that spindlin1 largely as a in solution (Fig. Thr-95 an for phosphate by in the loop between I and were found in the refined structure of spindlin1 and named and and their in the overall structure is shown in with heavy atom as phosphorous anomalous electron density at the phosphate the of (Fig. that spindlin1 associates with phosphate in a for in phosphate and residues, but or residues, are located in the phosphate which form a The between and phosphate to the phosphate further the phosphate also form a of with spindlin1. are between and spindlin1. The of phosphate three two are with the and of and is by the water with the of The also three two with the and of and by water with The and of phosphate form two with the The and of form two with whereas the two are generated between and the of Thr-95 and (Fig. The length of is in Table between phosphate and spindlin in to the of the between water and in to the of the between water and in to the of the between water and in to the of the between water and in to the of the between water and in to the of the between water and in to the of the between water and in to the of the between water and in to the of the between water and in to the of the between water and in to the of the between water and in to the of the between water and in to the of the between water and in to the of the between water and in to the of the between water and in to the of the between water and The in to the of the between water and Open table in a new tab with the for the phosphate are with a of The of the of three two with the and of and by water with the O atom of The of two with of and of The with of whereas with of and by water with the atom of our structure, the long loop is (Fig. with an B factor of for are in this loop that with the phosphate and or by water molecules and the of phosphate and greatly the of this to the two phosphate ions, the with the phosphate atom are the as by the electron density between the phosphate atom and of Thr-95 at a of the in wild type spindlin1 is whereas 3 be for the T95A mutant, indicating a decrease of the phosphate in this mutant not be with the native crystallization which that the structure of spindlin1 is at least partially by the of Thr-95 is a residue for the of phosphate ions. The between I and the for Cell Cycle the of the phosphate on the function of spindlin1, the cell cycle distribution of HeLa cells were with fluorescent HeLa cells expressing wild type spindlin1 or the T95A cells were at for after with their and were to cell to the of of the cell cycle distribution of cells by that of cells transfected with wild type spindlin1 the whereas of HeLa cells and of cells transfected with were in indicating that of spindlin1 lead to a in the of cells in = The of cell analysis also showed a of cells spindlin1 was of spindlin1 wild type, of cells, and of cells transfected with vector, with a of = These were in with previous studies (10Gao Y. Yue W. Zhang P. Li L. Xie X. Yuan H. Chen L. Liu D. Yan F. Pei X. Biochem. Biophys. Res. Commun. 2005; 335: 343-350Crossref PubMed Scopus (39) Google Scholar, B.S. Dragstedt C. Notterpek L. Nolan G.P. Leukemia. 2002; 16: 1507-1518Crossref PubMed Scopus (38) Google Scholar). cells expressing the spindlin1 T95A mutant showed a decrease in the of cells in and phase with those expressing wild type spindlin1 and were to those of the cells are and (Fig. a and our data that Thr-95 is for the function of spindlin1. our structure the of the phosphate ions, which are by is to in the of the loop P. we that of Thr-95 the between the loop and and II, its the loop be related to the cell cycle regulation of spindlin1, further is to this by the that Tudor-like domains nucleic acid activity together with the cell cycle and functions of spindlin1, we to the of spindlin1 to nucleic acid. In this of different of protein and nucleic acids were on initial data showed that the of spindlin1 lead to a of the to of which suggests that spindlin1 with was not by the indicating that the association between spindlin1 and is similar using of as a not an that spindlin1 not (Fig. Furthermore, spindlin1 also for than for cycle as the than the cycle spindlin1 with (Fig. are found in the structure and the of not the are not to be for nucleic acid activity not data the of acid a basis for and the to the of this in further of not shown in the previous bioinformatics the spin/ssty repeats and domains similar spin/ssty domains are in a tandem repeat manner. a protein with is also expressed during and early A. K. Development. Google Scholar). It is for the cell of but an function T. P. 2004; PubMed Scopus Google Scholar). of observations between spin/ssty repeat and domains. has that the might be a protein a checkpoint gene for Y. O. P. 2004; PubMed Scopus Google that two tandem domains, was found to to J. B. V. E. R. B. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, B. A. C. F. J. B. E. Protein 2005; PubMed Scopus Google Scholar). Furthermore, the in a gene for is for its activity J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). of domains display a similar anti-parallel structure with spin/ssty repeats (Fig. and spindlin, the gene family that three spin/ssty might also a similar further is to other Tudor-like domains are also involved in nucleic acid on domains in showed that the between loop and loop are for whereas the and be for nucleic acid J. B. V. E. R. B. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). Although sequence showed that are largely in spin/ssty repeat as their are not the (Fig. b and and in spin/ssty repeat similar in the structure, whereas is located in a different and the of in an with of In and T. R. J. 2002; PubMed Scopus Google that the nucleic acid of Tudor-like in is composed by loop and loop and of and of that spindlin1 domains are greatly in loop in the loop (Fig. a and loop is in spin/ssty repeats than in other domains, to a between loop and loop It is not or not the variations to different spindlin1 with nucleic acid in a similar to other domains further The for the of is a protein from and and we in the crystal structure an between two spindlin1 molecules that from on our structure, the by association of the loop and B which in the of two that are not involved in These two associate with other in a as in the crystal. on we that spindlin1 has the to form studies that spindlin1 has a to form a a of can also be (Fig. the two sites are to be the sites for other spindlin1 are also found in other The of the phosphorylation in which is by of the residues, an asymmetric association similar to the spindlin1 and C. Chen Y. J. PubMed Scopus Google Scholar, Y. C. J. PubMed Scopus Google also that the two on the tandem repeats appear as an unit of further which be important for its is highly expressed at the and protein the maternal of the gene (1Oh B. Hwang S.Y. Solter D. Knowles B.B. Development. 1997; 124: 493-503PubMed Google Scholar), whereas its protein is found in and early and has been used as a protein for the cell cycle (2Oh B. Hampl A. Eppig J.J. Solter D. Knowles B.B. Mol. Reprod. Dev. 1998; 50: 240-249Crossref PubMed Scopus (37) Google Scholar, S.K. Cell. Full Text PDF PubMed Scopus Google Scholar, P. H. D. B. J. Cell Google Scholar). on the of spindlin1 to the cell and its for is that spindlin1 is in the which be important for its In we have the three-dimensional structure of spindlin1, which is the first to be from the spin/ssty gene family. The spindlin1 structure a novel arrangement of the repeat fold. Thr-95 is a important residue that roles in the of phosphate and the loop between I and and which is for cell cycle We have also identified nucleic acid as its a structural and biochemical basis for further of spindlin1. We for with structure and Chen and for with