Cofactor Hydrogen Bonding onto the Protein Main Chain Is Conserved in the Short Chain Dehydrogenase/Reductase Family and Contributes to Nicotinamide Orientation

Human estrogenic 17β-hydroxysteroid dehydrogenase (17β-HSD1), a member of the short chain dehydrogenase/reductase (SDR) family, is responsible for the biosynthesis of all active estrogens. The crystal structures of two C19-steroid ternary complexes (17β-HSD1-androstanedione-NADP and 17β-HSD1-androstenedione-NADP) reveal the critical role of Leu149 in regulating the substrate specificity and provide novel insight into the different fates of a conserved glutamate residue in the estrogen-specific proteins upon the binding of the keto and hydroxyl groups of steroids. The whole NADP molecule can be unambiguously defined in the NADP binary complex, whereas both ternary complexes show that the nicotinamide moiety of NADP cannot be located in the density maps. In both ternary complexes, the expected position of carboxamide oxygen of NADP is occupied by a water molecule, which makes a bifurcated hydrogen bond with the O3 of C19-steroid and the main chain nitrogen of Val188. These results demonstrate that the hydrogen bonding interaction between the main chain amide group and the carboxamide group of NAD(P)(H) plays an important role in anchoring the nicotinamide ring to the enzyme. This finding is substantiated by structural analyses of all 33 NAD(P)(H) complexes of different SDR proteins, because 29 structures of 33 show this interaction. This common feature reveals a general mechanism among the SDR family, providing a rational basis for inhibitor design against biologically relevant SDR targets. Human estrogenic 17β-hydroxysteroid dehydrogenase (17β-HSD1), a member of the short chain dehydrogenase/reductase (SDR) family, is responsible for the biosynthesis of all active estrogens. The crystal structures of two C19-steroid ternary complexes (17β-HSD1-androstanedione-NADP and 17β-HSD1-androstenedione-NADP) reveal the critical role of Leu149 in regulating the substrate specificity and provide novel insight into the different fates of a conserved glutamate residue in the estrogen-specific proteins upon the binding of the keto and hydroxyl groups of steroids. The whole NADP molecule can be unambiguously defined in the NADP binary complex, whereas both ternary complexes show that the nicotinamide moiety of NADP cannot be located in the density maps. In both ternary complexes, the expected position of carboxamide oxygen of NADP is occupied by a water molecule, which makes a bifurcated hydrogen bond with the O3 of C19-steroid and the main chain nitrogen of Val188. These results demonstrate that the hydrogen bonding interaction between the main chain amide group and the carboxamide group of NAD(P)(H) plays an important role in anchoring the nicotinamide ring to the enzyme. This finding is substantiated by structural analyses of all 33 NAD(P)(H) complexes of different SDR proteins, because 29 structures of 33 show this interaction. This common feature reveals a general mechanism among the SDR family, providing a rational basis for inhibitor design against biologically relevant SDR targets. Short chain dehydrogenases/reductases (SDR) 1The abbreviations used are: SDR, short chain dehydrogenase/reductase; 17β-HSD, 17β-hydroxysteroid dehydrogenase; E1, estrone; E2, 17β-estradiol; A-dione, androstanedione; 4-dione, androstenedione; DHT, dihydrotestosterone; NMN, nicotinamide mononucleotide; PEG, polyethylene glycol; RMSD, root mean-squared deviation. form a large, functionally heterogeneous protein family presently with about 3000 primary sequences deposited in databases and the corresponding genomes represent all forms of life (1Oppermann U. Filling C. Hult M. Shafqat N. Wu X. Lindh M. Shafqat J. Nordling E. Kallberg Y. Persson B. Jornvall H. Chem. Biol. Interact. 2003; 143-144: 247-253Crossref PubMed Scopus (547) Google Scholar). The enzymes in the SDR family span several EC classes, from oxidoreductases and lyases to isomerases, with oxidoreductases as the majority (1Oppermann U. Filling C. Hult M. Shafqat N. Wu X. Lindh M. Shafqat J. Nordling E. Kallberg Y. Persson B. Jornvall H. Chem. Biol. Interact. 2003; 143-144: 247-253Crossref PubMed Scopus (547) Google Scholar). As a member of the SDR family (2Jornvall H. Persson B. Krook M. Atrian S. Gonzalez-Duarte R. Jeffery J. Ghosh D. Biochemistry. 1995; 34: 6003-6013Crossref PubMed Scopus (1161) Google Scholar), human estrogenic 17β-hydroxysteroid dehydrogenase (17β-HSD1, EC.1.1.1.62) catalyzes the conversion of an inactive estrogen, estrone (E1), to the biologically active estrogen, 17β-estradiol (E2) (3Luu-The V. Mol. Endocrinol. 1989; 3: 1301-1309Crossref PubMed Scopus (276) Google Scholar). Breast cancer is one of the most common malignancies in women worldwide, and estrogens play an important role in the development of hormone-dependent breast cancer (4Vihko R. Apter D. CRC Crit. Rev. Oncol./Hematol. 1989; 9: 1-16Crossref PubMed Scopus (54) Google Scholar). This enzyme has been demonstrated to be involved in maintaining high E2 levels in breast tumors of postmenopausal women (5Poutanen M. Isomaa V. Peltoketo H. Vihko R. J. Steroid Biochem. Mol. Biol. 1995; 55: 525-532Crossref PubMed Scopus (98) Google Scholar, 6Miyoshi Y. Ando A. Shiba E. Taguchi T. Tamaki Y. Noguchi S. Int. J. Cancer. 2001; 94: 685-689Crossref PubMed Scopus (137) Google Scholar). Because E2 has a stimulatory effect on the proliferation of breast cancer cells (7Bonney R.C. Reed M.J. Beranek P.A. Ghilchik M.W. James V.H. J. Steroid Biochem. 1986; 24: 361-364Crossref PubMed Scopus (20) Google Scholar), blocking its formation by specific inhibition of 17β-HSD1 should be of paramount importance for the control of breast tumor growth. To develop new compounds capable to inhibit 17β-HSD1, a series of structure/function studies were conducted in several laboratories focusing on the understanding of steroid binding to the enzyme. The mechanism for estrogen (C18-steroid) recognition by 17β-HSD1 was previously studied using the crystallographic structure of the enzyme with estradiol (8Azzi A. Rehse P.H. Zhu D.W. Campbell R.L. Labrie F. Lin S.X. Nat. Struct. Biol. 1996; 3: 665-668Crossref PubMed Scopus (134) Google Scholar, 9Breton R. Housset D. Mazza C. Fontecilla-Camps J.C. Structure. 1996; 4: 905-915Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Despite the similar hydrophobicity existing between estrogens (C18-steorid) and androgens (C19-steroid), however, 17β-HSD1 exhibits a high substrate specificity and catalytic efficiency toward estrogens such as estrone (E1) but insignificant ability to catalyze the conversion of androgens (10Mendoza-Hernandez G. Calcagno M. Sanchez-Nuncio H.R. Diaz-Zagoya J.C. Biochem. Biophys. Res. Commun. 1984; 119: 83-87Crossref PubMed Scopus (11) Google Scholar, 11Gangloff A. Garneau A. Huang Y.W. Yang F. Lin S.X. Biochem. J. 2001; 356: 269-276Crossref PubMed Google Scholar). Recent investigation showed that androgens (C19-steroid) can bind in an alternative mode to the protein through a combined study of enzyme kinetics and x-ray crystallography (12Gangloff A. Shi R. Nahoum V. Lin S.X. FASEB J. 2003; 17: 274-276Crossref PubMed Scopus (84) Google Scholar). The original aim of our investigation is to further study the mechanism of steroid recognition and discrimination adopted by 17β-HSD1. Here we report the crystal structures of two ternary complexes: 17β-HSD1-androstanedione (A-dione)-NADP and 17β-HSD1-androstenedione (4-dione)-NADP, as well as the binary complex of 17β-HSD1-NADP. Surprisingly, these NADP complexes, combined with all available crystal structures of SDR family enzymes, reveal a critical interaction between the enzyme and the cofactor, which is highly conserved among SDR family enzymes. Purification and Crystallization Procedures—17β-HSD1 was purified from fresh human placenta according to a previously described rapid purification procedure (13Lin S.X. Yang F. Jin J.Z. Breton R. Zhu D.W. Luu-The V. Labrie F. J. Biol. Chem. 1992; 267: 16182-16187Abstract Full Text PDF PubMed Google Scholar, 14Yang F. Zhu D.W. Wang J.Y. Lin S.X. J. Chromatogr. 1992; 582: 71-76Crossref PubMed Scopus (19) Google Scholar). Protein concentration for the homogeneous 17β-HSD1 was determined using the Bradford assay. The protein sample was concentrated to 15∼17 mg/ml in a buffer containing 40 mm Tris, pH 7.5, 1 mm EDTA, 0.2 mm dithiothreitol, 20% glycerol, and 0.06% (w/v) β-OG. The optimized crystallization conditions were derived from previous results (15Zhu D.W. Lee X. Breton R. Ghosh D. Pangborn Lin S.X. J. Mol. Biol. PubMed Scopus Google Scholar). the ternary complex, the mm pH and 20% the complex, the mm pH 7.5, and 20% the NADP binary complex, the mm pH 7.5, and 20% 17β-HSD1 was using the in 1 and the were to the by several The concentration of was 1 NADP was to the concentration of mm and mm in the binary and ternary complex, The procedure and were from one crystal for complex using the crystal was through 1 were and for the ternary complex, the ternary complex and the NADP binary complex, The were using the PubMed Scopus Google Scholar). the with that of the complex structure previously determined structure were with from the complex the and The were using Biol. PubMed Scopus Google for and J.Y. M. A. PubMed Scopus Google for In a of the for and were into the and In the water was by using the PubMed Scopus Google and The of a was to be The in the and the chain were because density be The of the was with the M.W. J. Google Scholar). for all the structures in been deposited in the Protein with the and J. G. H. Res. PubMed Scopus Google for and binary ternary ternary of for the in is the from and the and for the with The is for a of which were in from of main for the in is the and the and for the with The is for a of which were in in a new and of SDR were from the Protein J. G. H. Res. PubMed Scopus Google Scholar). These structures were using the J.Y. M. A. PubMed Scopus Google Scholar). and in this investigation to group and one C19-steroid ternary complexes, and were and The binary complex was show Structure. 1996; 4: Full Text Full Text PDF PubMed Scopus Google and the of the can be from the in As in previous (8Azzi A. Rehse P.H. Zhu D.W. Campbell R.L. Labrie F. Lin S.X. Nat. Struct. Biol. 1996; 3: 665-668Crossref PubMed Scopus (134) Google Scholar, 9Breton R. Housset D. Mazza C. Fontecilla-Camps J.C. Structure. 1996; 4: 905-915Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, A. Shi R. Nahoum V. Lin S.X. FASEB J. 2003; 17: 274-276Crossref PubMed Scopus (84) Google Scholar, D. Zhu D.W. Pangborn Labrie F. Lin S.X. Structure. 1995; 3: Full Text Full Text PDF PubMed Scopus Google Scholar, M.W. M. T. Vihko Ghosh D. U. S. A. PubMed Scopus Google Scholar), density was for the of the protein and the between and in these The protein of these complexes similar to the previously with root between these complexes and the E2 complex (8Azzi A. Rehse P.H. Zhu D.W. Campbell R.L. Labrie F. Lin S.X. Nat. Struct. Biol. 1996; 3: 665-668Crossref PubMed Scopus (134) Google and for the of the NADP molecule is well defined in the density and the and of NADP similar to that of previously determined complex C. Breton R. Housset D. Fontecilla-Camps J.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). to the enzyme in an with the ring in an and the nicotinamide ring in a between NADP and the nicotinamide binding of 17β-HSD1 been described M.W. M. T. Vihko Ghosh D. U. S. A. PubMed Scopus Google Scholar, C. Breton R. Housset D. Fontecilla-Camps J.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the nicotinamide ring is through and The nicotinamide ring against the of and and its carboxamide group forms hydrogen with the amide group and the chain of In the hydrogen bond between the carboxamide group and the moiety to the of the bond in the the of the ring against the protein and the to the active density for the steroid was from the The of molecule from the Protein J. G. H. Res. PubMed Scopus Google Scholar, was to the density As in the complex, this C19-steroid the which from the binding mode of the in the of cannot well into the density all The density that the and located on the which is of a This that has been into in the of NADP the The previous studies that the group of can be to a group by 17β-HSD1 in the of NADP (10Mendoza-Hernandez G. Calcagno M. Sanchez-Nuncio H.R. Diaz-Zagoya J.C. Biochem. Biophys. Res. Commun. 1984; 119: 83-87Crossref PubMed Scopus (11) Google Scholar, 11Gangloff A. Garneau A. Huang Y.W. Yang F. Lin S.X. Biochem. J. 2001; 356: 269-276Crossref PubMed Google Scholar). was to the bind to the enzyme in a however, this the that a of can bind to the enzyme in the binding mode and be because of the of NADP in the enzyme the catalytic efficiency is with that for estrone the that all the can be into in the of NADP in the the the can the active and to the enzyme in a which is the molecule was on the of the molecule and with the of the to and in the of 17β-HSD1 with its the substrate recognition of the protein The of by hydrogen The hydrogen bonding interaction the O3 is the as that of the complex, that is to O3 makes a hydrogen bond with the main chain nitrogen of to the complex, the hydrogen bonding interaction between and is the group cannot a hydrogen bond with the group of because of the of a hydrogen As a of the between the chain of to provide for a water molecule which is the hydrogen bonding of the of of of and the between and the group of of the in the binding of A-dione, and estradiol complexes reveals on the for these for the two and the steroid binding of and complexes the and located in similar The in the binding show with the NADP binary In the density is density for the and the of the NADP the density corresponding to the nicotinamide moiety of NADP molecule is defined in the density The of a density in this to the that the nicotinamide and its in the these were from further water molecule the position of the carboxamide oxygen of the NADP molecule in the binary complex, is well defined because of its density that of the previously described NADP binary complex, the density for the group to the is This is to the that the chain of has to the protein and is of the hydrogen bonding of the The of the interaction by the which is in the To for the the of these is to and that of the group to because of its in the density maps. for the well defined of NADP with from 29 to The for these is with that of all protein structure of the ternary complex high to that of the complex and the C19-steroid molecule the as The density for the steroid that has been to that has been into crystallization to the of DHT, a of can bind to the enzyme with its group the catalytic and be into 4-dione, which is by the previous studies (10Mendoza-Hernandez G. Calcagno M. Sanchez-Nuncio H.R. Diaz-Zagoya J.C. Biochem. Biophys. Res. Commun. 1984; 119: 83-87Crossref PubMed Scopus (11) Google Scholar). the active to the enzyme in a as in the ternary the previous binary complex structure unambiguously showed that in a the this is to the because a of substrate in a different be in the x-ray structure to the catalytic The molecule the position as in the complex with a the the of the oxygen The group of the molecule, to of and A-dione, is to the main chain of by a molecule In the the complex, the complex show the of a water molecule between the and the To the from the of 4-dione, the of about with that of the the hydrogen bonding interaction by the of the hydrogen bonding the chain as by its with that of the complex the similar for the complex 33 for the the high of the molecule with that of and be by the of hydrogen bonding interaction with because both and hydrogen to a can be for the binding between the complex and the we to the moiety of the NADP molecule in the density maps. was to the of the The of two in this in the the moiety in that of the SDR of crystal structures been determined and deposited in the Protein for of SDR family of structural of the SDR family and the enzyme in different is among which 33 of complexes with the NAD(P)(H) J. G. H. Res. PubMed Scopus Google Scholar). The corresponding for these 33 NAD(P)(H) complexes as and of these 33 structures reveals that as as 29 proteins of the the the common binding interaction with the carboxamide group of the nicotinamide these 29 in the of SDR family proteins and demonstrate high in and of The carboxamide group of NAD(P)(H) plays an important role in the specific of the nicotinamide ring to enzyme catalytic As for the binary complex in which the amide group of forms a hydrogen bond with the carboxamide group of a main chain amide in all of these 29 proteins and which is located the binding of the nicotinamide as a hydrogen to the carboxamide the binding of the this main chain amide to one specific residue In the corresponding from as as to as as of 29 and The in the enzymes of and this the residue in the of from the previous residue is conserved in of These proteins a common residue this which is in of containing crystal to form a bifurcated hydrogen bond with the carboxamide group and one oxygen of the To the recognition and discrimination of by 17β-HSD1, been different enzyme and x-ray with all the available complexes, our demonstrate that between the steroid and the to the binding of steroids. was that was to bind in the alternative binding mode to the with the chain of Leu149 (12Gangloff A. Shi R. Nahoum V. Lin S.X. FASEB J. 2003; 17: 274-276Crossref PubMed Scopus (84) Google Scholar). we that two and 4-dione, can be in the alternative mode in the of the active for all available crystal structures of 17β-HSD1 reveals that the steroid binding is and that been for the in the binding in the of different steroids. the of of the has the C19-steroid to its position as to the of the results provide further that 17β-HSD1 by Leu149 with the group of androgens to substrate In by a critical residue be a general regulating substrate specificity of enzymes using as in the studies of enzymes and estrogen M. T. T. FASEB J. 1996; PubMed Scopus Google Scholar, Y. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The crystal structures with the previously determined E2 and complexes of 17β-HSD1, an of the of recognition and discrimination by the two C19-steroid complexes, in with the and complexes, provide novel for the estrogen-specific proteins on the critical residue to the hydroxyl and groups which of the of estrogens and in the substrate recognition is in all steroid complexes of 17β-HSD1, to be involved in hydrogen bonding interaction with the of the different fates been for important residue the recognition of the In the E2 complex, the hydroxyl of estradiol makes a hydrogen bond with the group of (8Azzi A. Rehse P.H. Zhu D.W. Campbell R.L. Labrie F. Lin S.X. Nat. Struct. Biol. 1996; 3: 665-668Crossref PubMed Scopus (134) Google Scholar). a interaction has been between and the group of an alternative binding mode in the complex (12Gangloff A. Shi R. Nahoum V. Lin S.X. FASEB J. 2003; 17: 274-276Crossref PubMed Scopus (84) Google Scholar). In to is for the two complexes, the between the group of and the group of the steroid in the structures in this report is because of the of all available crystal structures of estrogen-specific proteins that these proteins a common among which is a conserved glutamate residue as a that with the group of estrogens V. A. Shi R. Lin S.X. FASEB J. 2003; 17: PubMed Scopus Google Scholar). study on estrogen showed that plays a role in binding the group of estradiol and in discrimination between estrogens and androgens J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This interaction was further by the crystal structure of the complex T. M. PubMed Scopus Google Scholar). These that the of hydrogen bonding between and is an for the proteins to J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is to in the of the hydroxyl group of the estrogen is a hydrogen and the group of the corresponding glutamate residue is a hydrogen the in the the group of the is a hydrogen and the carboxamide group of is a hydrogen This was by of such as by the in the B. A. F. F. J. 2001; PubMed Scopus Google Scholar). The different of the group to the corresponding hydroxyl keto group of by our crystal the the the most from these structures in the binding of the NADP molecule in the of we determined the crystal structure of the binary complex which well defined density for the whole NADP molecule and similar as in the previous ternary complex C. Breton R. Housset D. Fontecilla-Camps J.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In both the and the complex, however, is to that the corresponding to be located in the density maps. that the nicotinamide moiety has been the procedure because the crystallization conditions for the binary and ternary complexes for the of C19-steroid in the of the density for the two C19-steroid ternary complexes reveals that one water molecule, by the main chain amide group of and the group of the the position of the carboxamide oxygen of binding of the This that the conserved water molecule is an of C19-steroid binding because we the of such water molecule in the binary complex (12Gangloff A. Shi R. Nahoum V. Lin S.X. FASEB J. 2003; 17: 274-276Crossref PubMed Scopus (84) Google This water molecule be involved in the procedure of we its to the and the that the binding of the the of the this water molecule the the to the main chain nitrogen of and the moiety of the to the of The and the of the to be further the moiety of NADP in the ternary complexes occupied the position as in the NADP binary is to the carboxamide oxygen is the hydrogen bond of the main chain amide of Val188. In this the between the carboxamide oxygen of NADP and the group of the C19-steroid be to an because of the of hydrogen for both the carboxamide group of NADP to the with the of This is that the carboxamide nitrogen can a hydrogen bond with the group of the carboxamide oxygen be in a position to the oxygen As in the NADP binary complex, the hydrogen bonding interaction between NADP and the main chain amide group of the as well as the hydrogen bond between the carboxamide nitrogen and the both important to the nicotinamide ring in the as be expected for a enzyme. the binding of the C19-steroid and of the whole NADP is this as a of the with the O3 of the carboxamide group of NADP in the ternary complexes has to be in that of the from the main chain amide this for the to form the hydrogen bond between its carboxamide and as in the NADP binary complex, these specific for the enzyme to bind the NADP nicotinamide moiety the As a the with the O3 of C19-steroid results in the of the which in the NADP in the ternary that because the binding of the moiety in the NADP binary complex is from the of we that the of NADP in the ternary complex from the expected binding as in the NADP binary complex and to the is that the moiety of NADP because of the of the analyses for all available crystal structures of the SDR enzymes reveal that the interaction between a main chain amide group and the carboxamide group of is highly conserved in this Despite all available crystal structures for this family highly similar with a of the conserved located in the moiety binding and to be involved in the interaction with the carboxamide group is in structures H. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar, C. J. S. T. Nordling E. R. Jornvall H. U. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of structures the interaction between this residue and the In of the 33 structures reveal an of hydrogen bonding interaction between a main chain located the of and the carboxamide oxygen of cofactor, of the of in 29 of 33 This finding cannot be through in to the for these enzymes. The is the high of the residue involved in this as by the that this residue is of as as different This interaction can a high of and can be because the group is the main the an the important role of this interaction for the and of in the SDR this position a for This from the that the nicotinamide ring can its by against the The interaction the main chain amide group cannot presently be in the structures and for in which the binding for the moiety is most the interaction show upon the binding of substrate in the enzymes involved in the complex The determined structure of a in our structural analyses and which the conserved interaction we that this protein the SDR the of SDR J. S. A. J. 2001; PubMed Scopus Google Scholar). The of SDR enzymes SDR as (1Oppermann U. Filling C. Hult M. Shafqat N. Wu X. Lindh M. Shafqat J. Nordling E. Kallberg Y. Persson B. Jornvall H. Chem. Biol. Interact. 2003; 143-144: 247-253Crossref PubMed Scopus (547) Google Scholar). among the to the SDR family, for rational design for the and control of and is for design for the of several and study showed that two highly can the binding and the moiety in M. D. R.L. J. Chem. 2001; PubMed Scopus Google Scholar). in a member of the SDR family, the chain of the makes a hydrogen bond with the hydroxyl group of the conserved residue and the position of the nicotinamide ring of the D. M. Pangborn Structure. Full Text Full Text PDF PubMed Scopus Google Scholar). the that the amide group is important for the and of in the SDR family and is in the of substrate binding specific be to with the as well as to the binding of in enzymes. our finding on the conserved interaction between the and the main chain amide group in SDR our understanding on the for this family the development of against biologically relevant SDR targets. G. J. for critical and G. for the of the

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