Solution Structure of Selenoprotein W and NMR Analysis of Its Interaction with 14-3-3 Proteins

Selenium is a trace element with significant biomedical potential. It is essential in mammals due to its occurrence in several proteins in the form of selenocysteine (Sec). One of the most abundant mammalian Sec-containing proteins is selenoprotein W (SelW). This protein of unknown function has a broad expression pattern and contains a candidate CXXU (where U represents Sec) redox motif. Here, we report the solution structure of the Sec13 → Cys variant of mouse SelW determined through high resolution NMR spectroscopy. The protein has a thioredoxin-like fold with the CXXU motif located in an exposed loop similarly to the redox-active site in thioredoxin. Protein dynamics studies revealed the rigidity of the protein backbone and mobility of two external loops and suggested a role of these loops in interaction with SelW partners. Molecular modeling of structures of other members of the Rdx family based on the SelW structure identified new conserved features in these proteins, including an aromatic cluster and interacting loops. Our previous study suggested an interaction between SelW and 14-3-3 proteins. In the present work, with the aid of NMR spectroscopy, we demonstrated specificity of this interaction and identified mobile loops in SelW as interacting surfaces. This finding suggests that 14-3-3 are redox-regulated proteins. Selenium is a trace element with significant biomedical potential. It is essential in mammals due to its occurrence in several proteins in the form of selenocysteine (Sec). One of the most abundant mammalian Sec-containing proteins is selenoprotein W (SelW). This protein of unknown function has a broad expression pattern and contains a candidate CXXU (where U represents Sec) redox motif. Here, we report the solution structure of the Sec13 → Cys variant of mouse SelW determined through high resolution NMR spectroscopy. The protein has a thioredoxin-like fold with the CXXU motif located in an exposed loop similarly to the redox-active site in thioredoxin. Protein dynamics studies revealed the rigidity of the protein backbone and mobility of two external loops and suggested a role of these loops in interaction with SelW partners. Molecular modeling of structures of other members of the Rdx family based on the SelW structure identified new conserved features in these proteins, including an aromatic cluster and interacting loops. Our previous study suggested an interaction between SelW and 14-3-3 proteins. In the present work, with the aid of NMR spectroscopy, we demonstrated specificity of this interaction and identified mobile loops in SelW as interacting surfaces. This finding suggests that 14-3-3 are redox-regulated proteins. Many cellular processes are known to be regulated through reduction and oxidation (redox) (1Halliwell B. Plant Physiol. 2006; 141: 312-322Crossref PubMed Scopus (1574) Google Scholar). This regulation is carried out by a set of designated proteins, oxidoreductases. The redox function of these proteins may be supported by protein-bound cofactors or involve redox-active cysteine residues. Previous research on thiol oxidoreductases has focused only on a few of these enzymes (2Lillig C.H. Antioxid. Redox Signal. 2007; 9: 25-47Crossref PubMed Scopus (589) Google Scholar). Most prominent among them are proteins of the thioredoxin fold superfamily, such as thioredoxin, glutaredoxin, peroxiredoxin, glutathione peroxidase, and their more distant homologs (3Martin J.L. Structure. 1995; 3: 245-250Abstract Full Text Full Text PDF PubMed Scopus (691) Google Scholar). Thiol oxidoreductases, however, are much more widespread, and most members of this protein class have not been functionally characterized (4Fomenko D.E. Xing W. Adair B.M. Thomas D.J. Gladyshev V.N. Science. 2007; 315: 387-389Crossref PubMed Scopus (160) Google Scholar). In addition to oxidoreductases containing catalytic redox-active thiols, some enzymes evolved that possess an active site selenocysteine (Sec) 2The abbreviations used are:SecselenocysteineNOEnuclear Overhauser effectNOESYNOE spectroscopy. 2The abbreviations used are:SecselenocysteineNOEnuclear Overhauser effectNOESYNOE spectroscopy. (5Stadtman T.C. Annu. Rev. Biochem. 1996; 65: 83-100Crossref PubMed Scopus (811) Google Scholar). Sec is known as the 21st amino acid in proteins (6Hatfield D.L. Berry M. Gladyshev V.N. Selenium: Its Molecular Biology and Role in Human Health. Springer-Verlag New York Inc., New York2006Crossref Scopus (22) Google Scholar). This rare amino acid is inserted into proteins contranslationally in response to a UGA codon. The human set of Sec-containing proteins (selenoproteomes) consists of 25 known members (7Kryukov G.V. Castellano S. Novoselov S.V. Lobanov A.V. Zehtab O. Guigo R. Gladyshev V.N. Science. 2003; 300: 1439-1443Crossref PubMed Scopus (1839) Google Scholar), and selenoproteomes have also been analyzed in a variety of other organisms. Several selenoproteins have recently been functionally characterized and found to be oxidoreductases (8Flohe L. Dev. Ophthalmol. 2005; 38: 89-102PubMed Google Scholar). For example, both major cellular redox systems, the thioredoxin and glutathione systems, are dependent on selenoproteins (thioredoxin reductases and glutathione peroxidases, respectively). However, the functions of the majority of selenoproteins are not known. selenocysteine nuclear Overhauser effect NOE spectroscopy. selenocysteine nuclear Overhauser effect NOE spectroscopy. We recently reported on a family of selenoproteins and their Cys homologs, designated as the Rdx family (9Dikiy A. Novoselov S.V. Fomenko D.E. Sengupta A. Carlson B.A. Cerny R.L. Ginalski K. Grishin N.V. Hatfield D.L. Gladyshev V.N. Biochemistry. 2007; 46: 6871-6882Crossref PubMed Scopus (177) Google Scholar). Mammalian members of this family include selenoproteins SelW, SelT, SelH, SelV, and a Cys-containing Rdx12 protein. Functions are not known for any of these proteins. Through sequence analysis and structure modeling, we predicted that these proteins possess a thioredoxin-like fold, suggesting a redox nature of catalytic Sec residues. Among Rdx proteins, the best characterized is SelW. It is a 9-kDa selenoprotein, abundant in muscle and brain (10Beilstein M.A. Vendeland S.C. Barofsky E. Jensen O.N. Whanger P.D. J. Inorg. Biochem. 1996; 61: 117-124Crossref PubMed Scopus (100) Google Scholar, 11Vendeland S.C. Beilstein M.A. Chen C.L. Jensen O.N. Barofsky E. Whanger P.D. J. Biol. Chem. 1993; 268: 17103-17107Abstract Full Text PDF PubMed Google Scholar, 12Gu Q.P. Sun Y. Ream L.W. Whanger P.D. Mol. Cell Biochem. 2000; 204: 49-56Crossref PubMed Google Scholar, 13Sun Y. Gu Q.P. Whanger P.D. J. Inorg. Biochem. 2001; 84: 151-156Crossref PubMed Scopus (24) Google Scholar, 14Loflin J. Lopez N. Whanger P.D. Kioussi C. J. Inorg. Biochem. 2006; 100: 1679-1684Crossref PubMed Scopus (98) Google Scholar). Expression of SelW is regulated by the availability of selenium in the diet. Native SelW was reported to occur in a complex with glutathione, which was attached to one of the Cys residues in the protein (10Beilstein M.A. Vendeland S.C. Barofsky E. Jensen O.N. Whanger P.D. J. Inorg. Biochem. 1996; 61: 117-124Crossref PubMed Scopus (100) Google Scholar). Very little is known about SelT and Rdx12 (9Dikiy A. Novoselov S.V. Fomenko D.E. Sengupta A. Carlson B.A. Cerny R.L. Ginalski K. Grishin N.V. Hatfield D.L. Gladyshev V.N. Biochemistry. 2007; 46: 6871-6882Crossref PubMed Scopus (177) Google Scholar), whereas recent studies revealed that SelH is a nucleolar protein that protects cells against stress associated with hydrogen peroxide (15Novoselov S.V. Kryukov G.V. Xu X.M. Carlson B.A. Hatfield D.L. Gladyshev V.N. J. Biol. Chem. 2007; 282: 11960-11968Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). SelV is highly homologous to SelW but has an additional N-terminal proline-rich extension and is expressed specifically in testes (7Kryukov G.V. Castellano S. Novoselov S.V. Lobanov A.V. Zehtab O. Guigo R. Gladyshev V.N. Science. 2003; 300: 1439-1443Crossref PubMed Scopus (1839) Google Scholar). By utilizing affinity columns containing mutant versions of Rdx proteins, we found that SelW binds 14-3-3 proteins, whereas Rdx12 binds glutathione peroxidase 1 (9Dikiy A. Novoselov S.V. Fomenko D.E. Sengupta A. Carlson B.A. Cerny R.L. Ginalski K. Grishin N.V. Hatfield D.L. Gladyshev V.N. Biochemistry. 2007; 46: 6871-6882Crossref PubMed Scopus (177) Google Scholar). The functional importance of these interactions is not known. The SelW/14-3-3 interaction is particularly interesting, since the latter proteins are a class of abundant proteins with multiple cellular functions and roles in disease (16Yaffe M.B. FEBS Lett. 2002; 513: 53-57Crossref PubMed Scopus (555) Google Scholar, 17van Heusden G.P.H. IUBMB Life. 2005; 57: 623-629Crossref PubMed Scopus (107) Google Scholar, 18Darling D.L. Yingling J. Wynshaw-Boris A. Curr. Top. Dev. Biol. 2005; 68: 281-315Crossref PubMed Scopus (129) Google Scholar, 19Pozuelo Rubio M. Geraghty K.M. Wong B.H. Wood N.T. Campbell D.G. Morrice N. Mackintosh C. Biochem. J. 2004; 379: 395-408Crossref PubMed Scopus (382) Google Scholar). In addition, the SelW/14-3-3 interaction differs from that observed for other 14-3-3 partners, since SelW does not have canonical 14-3-3 binding motifs (R(S/X)XpSXPorRXXXpSXP, where X denotes any amino acid residue, and pS represents a phosphorylated serine (20Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Gamblin S.J. Smerdon S.J. Cantley L.C. Cell. 1997; 91: 961-971Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar, 21Rittinger K. Budman J. Xu J. Volinia S. Cantley L.C. Smerdon S.J. Gamblin S.J. Yaffe M.B. Mol. Cell. 1999; 4: 153-166Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar). One possibility is that SelW regulates 14-3-3 in a redox manner; in the oxidized form (e.g. with a disulfide or a glutathionylated cysteine), 14-3-3 may be inactive and require a reduced form of SelW for activation. Determination of the three-dimensional structure of SelW as well as structural information on the SelW/14-3-3 interaction would be key in understanding both the structural basis and biological relevance of this interaction. NMR spectroscopy is a powerful technique for structural characterization of proteins in solution as well as for monitoring interactions between two or more proteins. Additionally, NMR provides valuable information regarding mobility of various parts of the protein and its residues. Here, we used high resolution NMR spectroscopy to determine the structure of a founding member of the Rdx family, SelW, and examined its interactions with 14-3-3 proteins. These data revealed the thioredoxin-like fold in SelW and, therefore, other Rdx proteins and provided structural insights into the interaction of SelW with 14-3-3. Expression and Purification of SelW—Structural analysis was carried out with a recombinant bacterially expressed mouse SelW tagged at the C terminus with a His6 tag and containing Cys in place of Sec13 and Ser in place of Cys10 (9Dikiy A. Novoselov S.V. Fomenko D.E. Sengupta A. Carlson B.A. Cerny R.L. Ginalski K. Grishin N.V. Hatfield D.L. Gladyshev V.N. Biochemistry. 2007; 46: 6871-6882Crossref PubMed Scopus (177) Google Scholar). To uniformly label SelW with 15Nor 15N/13C, cells were grown in M9 minimal medium containing 99% enriched (15NH4)2SO4 and 98% enriched 13C6-labeled d-glucose (Spectra Stable Isotopes). His6-SelW was overexpressed in Escherichia coli ER2566 (New England Biolabs) by growing cells at 37 °C until the A600 nm reached 0.6, followed by induction of protein synthesis by the addition of 1 mm isopropyl 1-thio-β-d-galactopyranoside for 3 h. Cells were collected by centrifugation and disrupted by sonication in 25 mm phosphate buffer, pH 7.0, containing 10 mm NaCl and 0.05% Triton X-100 (Sigma). Additionally, 1 mm lysozyme (Sigma) and a half-tablet of Complete Protease Inhibitor mixture (Roche Applied Science) were added to the solution. Following centrifugation, supernatant was applied onto a TALON Co-IMAC-Sepharose column (BD Biosciences). The column was washed with 20 mm imidazole in mm phosphate buffer, pH 7.0, mm followed by of protein with mm imidazole in mm phosphate buffer, pH 7.0, mm NaCl at Expression and Purification of expression that for N-terminal with the provided by A. and and from and provided by The were into E. coli ER2566 (New England 14-3-3 proteins were by growing cells in 14-3-3 were overexpressed by growing cells at 37 °C to A600 nm 0.6, followed by induction of protein synthesis with 1 mm isopropyl 1-thio-β-d-galactopyranoside for h. Cells containing N-terminal were collected by centrifugation and disrupted containing Complete Protease Inhibitor mixture (Roche Applied Following centrifugation, the supernatant was applied onto an acid column The column was washed with 20 mm imidazole in mm phosphate buffer, pH 7.0, mm NaCl buffer, followed by protein with mm imidazole in at Cells tagged were by disrupted by sonication in mm buffer, pH containing mm mm and and with 1 mm lysozyme (Sigma) and Complete mixture (Roche Applied Cell was applied onto a acid column The column was washed with 25 mm imidazole in mm buffer, pH containing mm NaCl and followed by of protein with mm imidazole in mm buffer, pH containing mm NaCl at for NMR studies mm His6-SelW in 25 mm phosphate buffer, pH with mm mm in the of or of SelW with between SelW and 14-3-3 was by mm SelW with 14-3-3 at a of in 25 mm phosphate buffer, pH containing mm were and the were as (Sigma) was with SelW as for and the were NMR NMR were at or on a with a and were to of acid as in S. J. 2003; PubMed Scopus Google backbone and of SelW were and were from and NOE were from three-dimensional and were from three-dimensional NMR data were with the and analysis was R. the of and of Scholar). were from two and sequence R. Biochemistry. PubMed Scopus Google as the of the with and and of were by of in with R. Biochemistry. PubMed Scopus Google Scholar, A. Biochemistry. PubMed Scopus Google Scholar). NMR data reported in this were collected at the of and of and NMR were and in the the R. the of and of Scholar). The M. W. K. J. Mol. Biol. PubMed Scopus Google in was used to from into were from three-dimensional and were and with backbone from the A. J. 1999; PubMed Scopus Google Scholar). the basis of this the structure was the dynamics C. K. J. Mol. Biol. 1997; PubMed Scopus Google Scholar). from with and the 20 with the function were in a the K.M. J. Chem. 1995; Scopus Google with the aid of the of Rdx of mouse Rdx12 and SelT were based on the NMR structure of mouse SelW. of the SelW of and for a recombinant and variant of mouse SelW was through and three-dimensional NMR The was at two and K. of NMR that in the with the were in the were from and three-dimensional and 1 of among to the of and were from three-dimensional and were and the A. J. 1999; PubMed Scopus Google Scholar). In NOE and were used to the three-dimensional structure of SelW the high of the The structures the family were to the K.M. J. Chem. 1995; Scopus Google Scholar). The of the family with the were in the Protein of NMR data and structural for the solution structure of the reduced recombinant Sec13 → Cys form of mouse data for structure NOE 20 are the and the 20 with the function that the NMR solution family function in in in in to the The are the and the 20 with the function that the NMR solution in a new 1 a family of 20 and of the three-dimensional structure and of SelW. for the structure is reported in The data that the SelW structure represents a with the structural data well the found The observed structural family is well backbone with to the structure is only the protein characterized by the of structure are the to the in the SelW family are located in two external loops resolution for these is due to a of for residues the two loops. One of the loops contains a predicted active site of SelW (e.g. the CXXU whereas the loop may be in binding The structural resolution observed for the two loops may be a of mobility of these with to the of the protein. the mobility may have a role may be for binding between interacting To this characterization of the protein by NMR spectroscopy was carried out The structure of SelW consists of a with two and a located on one of the and these the protein contains two external loops and a The is characterized by a structure and are a which is also observed in thioredoxin-like fold proteins Y. Grishin N.V. 2005; PubMed Scopus Google Scholar). The of is at an with to the of the as a of the The CXXU motif is located in the loop between and The loop and and form a with a in In the is out as observed for in W. K. J. Mol. Biol. 204: PubMed Scopus Google Scholar). of mouse SelW is in The data that are the protein the or a is found in the external loops and by in amino the of Cys in the CXXU This suggests that the interaction between SelW and its place the of be that of the loop aromatic amino as for the loops in to the protein backbone these residues are by and and a serine which backbone and loop these residues may both rigidity to the structural and to the in an structural for the predicted catalytic site of SelW. the two external loops in the solution structure of SelW a resolution with to the of the protein that be by loop To this we of SelW in a through analysis of backbone and and NOE these as a function of It is that the majority of residues and NOE of amino in a protein of the SelW However, the data the residues of the two external loops residues and of the in are with to of other therefore, the for and are The of the in suggests that the loop residues are more mobile the of the For the of SelW, the was a of the to an A. Biochemistry. PubMed Scopus Google Scholar). of and SelW the of this an structure of a distant SelW in the Protein of mammalian and proteins is from both functional and of of these two Protein and in amino acid in and not these proteins as homologs with mouse SelW and proteins an of both structure and the predicted active site motif. These proteins also the in mouse as well as residues in the of mouse and SelW structures are in 3 analysis was the of one SelW in the structure of the The backbone was three-dimensional structures in mammalian and In structural suggests that the proteins may functions in and the of aromatic residues in the mouse SelW aromatic and form a cluster the predicted active site of SelW. This cluster is from the motif is to have a role in the redox However, be for binding of interaction the protein This possibility is with the of aromatic residues in the also of mouse and SelW of Rdx three-dimensional structure of SelW to structures of other members of the Rdx SelT, and SelW with the structure of reduced thioredoxin The proteins several structural including the structure However, in thioredoxin, an additional is inserted between the and This is of the thioredoxin Rdx family proteins possess a thioredoxin-like fold as recently Y. Grishin N.V. 2005; PubMed Scopus Google Scholar). The Rdx12 structural is to the structure of SelW, with the that Rdx12 has a acid N-terminal extension of the as well as amino of The structure of and that of SelW. in the motif to be in Rdx12 in SelW, and its is such that the N-terminal of the protein. The SelT structure The N-terminal with the in in the motif with to SelW. Additionally, SelT contains a acid of predicted between and The function of this is not but is located in the structure the CXXU motif. Our structural analysis that the predicted active of SelW, and SelT are located in the external loop between and this structural element in Rdx proteins is the aromatic cluster the active suggesting a function of this cluster in these proteins. Additionally, two external loops in this class of proteins residues. of the Rdx and Sec-containing the structures of Cys versions of two thioredoxin-like and were determined Fomenko D.E. Y. J. Gladyshev V.N. J. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google and respectively). These proteins are to Rdx proteins in an thioredoxin-like fold and the thioredoxin the structural motif. The loop to be in and to that in the Rdx One between Rdx proteins on one and and on the other is that the in the latter proteins is into two with a in proteins in and residues the two external which to be a of these proteins. the other thioredoxin, and in to Rdx proteins, not have the aromatic the fold of of these proteins is the we the role of aromatic residues in protein between SelW and previous studies revealed that 14-3-3 proteins SelW in and (9Dikiy A. Novoselov S.V. Fomenko D.E. Sengupta A. Carlson B.A. Cerny R.L. Ginalski K. Grishin N.V. Hatfield D.L. Gladyshev V.N. Biochemistry. 2007; 46: 6871-6882Crossref PubMed Scopus (177) Google Scholar). To the structural basis of this we examined binding between these two proteins by NMR spectroscopy. The interaction between SelW and 14-3-3 was through in of SelW the addition of 14-3-3. This the with the of SelW were not by additional from 14-3-3 and be and of 14-3-3 were as both and proteins. 14-3-3 containing a tag was the protein containing an N-terminal tag was not and, therefore, was not for structural with this the of SelW was not the addition of the tagged that the 14-3-3 does not with SelW. In the addition of or of 14-3-3 containing to the SelW in the significant in the SelW of SelW and SelW in the of the are in and that the pattern of SelW in both the the significant from the SelW the addition of 14-3-3 in the and other whereas the of the majority of the in the NMR are of interaction between these proteins. Our therefore, previous data (9Dikiy A. Novoselov S.V. Fomenko D.E. Sengupta A. Carlson B.A. Cerny R.L. Ginalski K. Grishin N.V. Hatfield D.L. Gladyshev V.N. Biochemistry. 2007; 46: 6871-6882Crossref PubMed Scopus (177) Google on the interaction between 14-3-3 and SelW. The that SelW does not with the tagged 14-3-3 that the observed interaction between SelW and 14-3-3 is To this we in SelW the addition of The latter is a protein and has a with that of 14-3-3 is a 1 the of SelW in the of at a of of this with that of SelW identified that these two proteins not in solution. data that the interaction is both and of the SelW NMR we also determined the of SelW residues that are in this interaction by of the in the the addition of the in or of NMR are in the interaction between two whereas other for which are are to in M. K. Rev. 2002; PubMed Scopus Google Scholar, Chem. 2004; PubMed Scopus Google Scholar). In work, both of and of NMR were observed the addition of to SelW. for the as a function of The for residues in the and therefore, were located at the interacting was to reported in for The was also observed for be in SelW contains characterized by of NMR of amino in the interaction with residues and These are on the SelW structure and in Our data that two external loops and and a of SelW are in binding of 14-3-3. We that the mobility of these loops is functionally and is for interaction of SelW with its partners. Our reported regarding this interaction through studies (9Dikiy A. Novoselov S.V. Fomenko D.E. Sengupta A. Carlson B.A. Cerny R.L. Ginalski K. Grishin N.V. Hatfield D.L. Gladyshev V.N. Biochemistry. 2007; 46: 6871-6882Crossref PubMed Scopus (177) Google Scholar). that SelW and 14-3-3 proteins in solution the of an the thioredoxin-like fold and motif in SelW, such interaction be The most for this would involve reduction of the oxidized 14-3-3 protein by SelW. that the is located in the external loop and the role of the in the reduction whereas Cys10 may be a The of the interaction between SelW and 14-3-3 of the structure of the in in this study the solution structure of mammalian SelW that was through high resolution NMR spectroscopy. The structure revealed a thioredoxin-like fold, a structure in the motif in an exposed loop in a to the redox-active site in thioredoxin. The SelW structure of the of homologous proteins and of their The structure also suggested a role for SelW in redox of protein dynamics revealed rigidity of the backbone for two external loops. These loops to in the interaction of SelW with 14-3-3 proteins. interaction with 14-3-3 protein was found to involve the conserved active site CXXU suggesting that the interaction on the redox of SelW. We A. Aitken of for an expression for the N-terminal and of for the 14-3-3 proteins. We are to for with and with