The Crystal Structure of Progesterone 5β-Reductase from Digitalis lanata Defines a Novel Class of Short Chain Dehydrogenases/Reductases
2007; Elsevier BV; Volume: 283; Issue: 25 Linguagem: Inglês
10.1074/jbc.m706185200
ISSN1083-351X
AutoresAndrea Thorn, C. Egerer-Sieber, Christof M. Jäger, Vanessa Herl, Frieder Müller‐Uri, Wolfgang Kreis, Yves A. Muller,
Tópico(s)Plant biochemistry and biosynthesis
ResumoProgesterone 5β-reductase (5β-POR) catalyzes the stereospecific reduction of progesterone to 5β-pregnane-3,20-dione and is a key enzyme in the biosynthetic pathway of cardenolides in Digitalis (foxglove) plants. Sequence considerations suggested that 5β-POR is a member of the short chain dehydrogenase/reductase (SDR) family of proteins but at the same time revealed that the sequence motifs that in standard SDRs contain the catalytically important residues are missing. Here we present crystal structures of 5β-POR from Digitalis lanata in complex with NADP+ at 2.3Å and without cofactor bound at 2.4Å resolution together with a model of a ternary complex consisting of 5β-POR, NADP+, and progesterone. Indeed, 5β-POR displays the fold of an extended SDR. The architecture of the active site is, however, unprecedented because none of the standard catalytic residues are structurally conserved. A tyrosine (Tyr-179) and a lysine residue (Lys-147) are present in the active site, but they are displayed from novel positions and are part of novel sequence motifs. Mutating Tyr-179 to either alanine or phenylalanine completely abolishes the enzymatic activity. We propose that the distinct topology reflects the fact that 5β-POR reduces a conjugated double bond in a steroid substrate via a 1–4 addition mechanism and that this requires a repositioning of the catalytically important residues. Our observation that the sequence motifs that line the active site are conserved in a number of bacterial and plant enzymes of yet unknown function leads us to the proposition that 5β-POR defines a novel class of SDRs. Progesterone 5β-reductase (5β-POR) catalyzes the stereospecific reduction of progesterone to 5β-pregnane-3,20-dione and is a key enzyme in the biosynthetic pathway of cardenolides in Digitalis (foxglove) plants. Sequence considerations suggested that 5β-POR is a member of the short chain dehydrogenase/reductase (SDR) family of proteins but at the same time revealed that the sequence motifs that in standard SDRs contain the catalytically important residues are missing. Here we present crystal structures of 5β-POR from Digitalis lanata in complex with NADP+ at 2.3Å and without cofactor bound at 2.4Å resolution together with a model of a ternary complex consisting of 5β-POR, NADP+, and progesterone. Indeed, 5β-POR displays the fold of an extended SDR. The architecture of the active site is, however, unprecedented because none of the standard catalytic residues are structurally conserved. A tyrosine (Tyr-179) and a lysine residue (Lys-147) are present in the active site, but they are displayed from novel positions and are part of novel sequence motifs. Mutating Tyr-179 to either alanine or phenylalanine completely abolishes the enzymatic activity. We propose that the distinct topology reflects the fact that 5β-POR reduces a conjugated double bond in a steroid substrate via a 1–4 addition mechanism and that this requires a repositioning of the catalytically important residues. Our observation that the sequence motifs that line the active site are conserved in a number of bacterial and plant enzymes of yet unknown function leads us to the proposition that 5β-POR defines a novel class of SDRs. The beneficial effects of cardenolides, also known as cardiac glycosides or cardiotonic steroids, are well documented, and they have been applied for the treatment of cardiac insufficiencies for centuries (1Withering W. Williams F.A. Keys T.E. Classics of Cardiology. Dover Publications, New York1941: 231-252Google Scholar, 2Gheorghiade M. Adams Jr., K.F. Colucci W.S. Circulation. 2004; 109: 2959-2964Crossref PubMed Scopus (187) Google Scholar, 3Antman E.M. Smith T.W. Annu. Rev. Med. 1985; 36: 357-367Crossref PubMed Scopus (26) Google Scholar). On a molecular level, these steroids are potent inhibitors of the sodium/potassium pump (Na+/K+-ATPase) that is present in almost all cells in higher organisms (4Mathews C.K. van Holde K.E. Ahern K.G. Biochemistry. 3rd Ed. Addison-Wesley Publishing Co., San Francisco2000: 340-342Google Scholar). Digitalis plants are still the major source for cardenolides, and as a step in the biosynthetic pathway, the Digitalis enzyme progesterone 5β-reductase (5β-POR) 2The abbreviations used are: 5β-POR, progesterone 5β-reductase; SDR, short chain dehydrogenase/reductase; AKR, aldo-keto-reductase; 17β-HSD, 17β-hydroxysteroid dehydrogenase; GMD, GDP mannose-4,6-dehydratase; HPLC, high pressure liquid chromatography; r.m.s., root mean square; PDB, Protein Data Bank. catalyzes the stereospecific NADPH-dependent reduction of the Δ4-double bond in progesterone to 5β-pregnane-3,20-dione. Because all Digitalis cardenolides share the characteristic 5β-configuration, the enzyme 5β-POR catalyzes a central step during their biosynthesis (5Herl V. Fischer G. Muller-Uri F. Kreis W. Phytochemistry. 2006; 67: 225-231Crossref PubMed Scopus (47) Google Scholar, 6Gärtner D.E. Wendroth S. Seitz H.U. FEBS Lett. 1990; 271: 239-242Crossref PubMed Scopus (45) Google Scholar, 7Caspi E. Lewis D.O. Science. 1967; 156: 519-520Crossref PubMed Scopus (40) Google Scholar). NADH/NADPH-dependent reductases as well as the related dehydrogenases, dehydratases, and epimerases can be classified into two major protein families: the (α/β)8-barrel containing aldo-keto-reductases (AKRs) (8Jez J.M. Penning T.M. Chem. Biol. Interact. 2001; 130–132: 499-525Crossref PubMed Scopus (217) Google Scholar) and the Rossman fold containing short chain dehydrogenases/reductases (SDRs) (9Rossman M.G. Liljas A. Branden C.-I. Banaszak L.J. Boyer P.D. The Enzymes. 3rd Ed. Academic Press, New York1975: 61-102Google Scholar, 10Kallberg Y. Oppermann U. Jornvall H. Persson B. Eur. J. Biochem. 2002; 269: 4409-4417Crossref PubMed Scopus (340) Google Scholar, 11Jornvall H. Persson B. Krook M. Atrian S. Gonzalez-Duarte R. Jeffery J. Ghosh D. Biochemistry. 1995; 34: 6003-6013Crossref PubMed Scopus (1159) Google Scholar). Additional families such as the long and medium chain dehydrogenases/reductases are related to SDRs because they share with the latter the dinucleotide-binding double Rossman fold (12Edwards K.J. Barton J.D. Rossjohn J. Thorn J.M. Taylor G.L. Ollis D.L. Arch. Biochem. Biophys. 1996; 328: 173-183Crossref PubMed Scopus (63) Google Scholar, 13Jornvall H. Hoog J.O. Persson B. FEBS Lett. 1999; 445: 261-264Crossref PubMed Scopus (172) Google Scholar, 14Jornvall H. Persson B. Jeffery J. Eur. J. Biochem. 1987; 167: 195-201Crossref PubMed Scopus (247) Google Scholar). SDRs are about 250 residues long and form a large family with over 2000 members (15Kallberg Y. Oppermann U. Jornvall H. Persson B. Protein Sci. 2002; 11: 636-641Crossref PubMed Scopus (197) Google Scholar). Because their central feature consists of an all-parallel β-sheet and their catalytic mechanism evolves around a tyrosine residue, they are also referred to as 7-stranded tyrosine-dependent oxidoreductases (16Murzin A.G. Brenner S.E. Hubbard T. Chothia C. J. Mol. Biol. 1995; 247: 536-540Crossref PubMed Scopus (5595) Google Scholar). The dinucleotide-binding double Rossman fold motif is contained within the N-terminal six β-strands of the seven-stranded β-sheet (strands βAto βG). Insertions of up to 100 residues in length occur in many SDRs and are predominantly accommodated within the left-handed crossover connection between strands βF and βG as well as after strand βG toward the C terminus of the protein. These two segments are often collectively referred to as the ligand-binding domain of SDRs, and they are considered the prime determinants of substrate specificity (17Webb N.A. Mulichak A.M. Lam J.S. Rocchetta H.L. Garavito R.M. Protein Sci. 2004; 13: 529-539Crossref PubMed Scopus (42) Google Scholar). The SDR family members can be identified at the sequence level based on several conserved motifs, and variations in these motifs have been used to define SDR subfamilies (15Kallberg Y. Oppermann U. Jornvall H. Persson B. Protein Sci. 2002; 11: 636-641Crossref PubMed Scopus (197) Google Scholar, 18Persson B. Kallberg Y. Oppermann U. Jornvall H. Chem. Biol. Interact. 2003; 143–144: 271-278Crossref PubMed Scopus (175) Google Scholar). These motifs are either involved in NADH/NADPH cofactor binding or cluster around the substrate-binding pocket. SDRs contain in their active site a highly conserved amino acid triad consisting of a serine, tyrosine, and lysine residue, with a possible fourth conserved asparagine residue (19Filling C. Berndt K.D. Benach J. Knapp S. Prozorovski T. Nordling E. Ladenstein R. Jornvall H. Oppermann U. J. Biol. Chem. 2002; 277: 25677-25684Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar). The conservation of the catalytic triad in almost all SDRs indicates that SDRs share a common reaction mechanism. Moreover, this mechanism seems also to extend to AKRs because they have the conserved tyrosine and lysine in common with SDRs (20Bennett M.J. Schlegel B.P. Jez J.M. Penning T.M. Lewis M. Biochemistry. 1996; 35: 10702-10711Crossref PubMed Scopus (104) Google Scholar). Based on sequence alignments, 5β-POR from Digitalis plants has been predicted to be an SDR family member (5Herl V. Fischer G. Muller-Uri F. Kreis W. Phytochemistry. 2006; 67: 225-231Crossref PubMed Scopus (47) Google Scholar, 21Gavidia I. Tarrio R. Rodriguez-Trelles F. Perez-Bermudez P. Seitz H.U. Phytochemistry. 2006; 68: 853-864Crossref PubMed Scopus (38) Google Scholar). This is in contrast to mammalian steroid 5β-reductase, which is a member of the AKR family (22Penning T.M. Ma H. Jez J.M. Chem. Biol. Interact. 2001; 130–132: 659-671Crossref PubMed Scopus (12) Google Scholar). Plant 5β-POR shares the typical NADPH/NADH-binding sequence motifs with other SDRs (18Persson B. Kallberg Y. Oppermann U. Jornvall H. Chem. Biol. Interact. 2003; 143–144: 271-278Crossref PubMed Scopus (175) Google Scholar), but intriguingly, none of the sequence motifs that cluster around the substrate-binding site and that contain the catalytically important residues are conserved. The sequence motif that displays the conserved active site serine residue in SDRs, namely GXXXXXSS (or SSXXXXG in some SDRs) (10Kallberg Y. Oppermann U. Jornvall H. Persson B. Eur. J. Biochem. 2002; 269: 4409-4417Crossref PubMed Scopus (340) Google Scholar, 18Persson B. Kallberg Y. Oppermann U. Jornvall H. Chem. Biol. Interact. 2003; 143–144: 271-278Crossref PubMed Scopus (175) Google Scholar), is missing in 5β-POR (5Herl V. Fischer G. Muller-Uri F. Kreis W. Phytochemistry. 2006; 67: 225-231Crossref PubMed Scopus (47) Google Scholar, 21Gavidia I. Tarrio R. Rodriguez-Trelles F. Perez-Bermudez P. Seitz H.U. Phytochemistry. 2006; 68: 853-864Crossref PubMed Scopus (38) Google Scholar). Also, the sequence motif YXXXK (or YXXMXXXK) (10Kallberg Y. Oppermann U. Jornvall H. Persson B. Eur. J. Biochem. 2002; 269: 4409-4417Crossref PubMed Scopus (340) Google Scholar, 18Persson B. Kallberg Y. Oppermann U. Jornvall H. Chem. Biol. Interact. 2003; 143–144: 271-278Crossref PubMed Scopus (175) Google Scholar) that displays the active site tyrosine and lysine residue is absent, and a conserved NFYYXXED motif can be found instead (5Herl V. Fischer G. Muller-Uri F. Kreis W. Phytochemistry. 2006; 67: 225-231Crossref PubMed Scopus (47) Google Scholar, 21Gavidia I. Tarrio R. Rodriguez-Trelles F. Perez-Bermudez P. Seitz H.U. Phytochemistry. 2006; 68: 853-864Crossref PubMed Scopus (38) Google Scholar). Although it has been suggested that one of the tyrosines in this motif corresponds to the typical SDR active site tyrosine, it is not possible to locate the additionally required lysine residue in any of the adjacent sequence segments. Hence, the question arises whether 5β-POR defines a novel class of SDRs with a different set of sequence motifs and conserved residues in the catalytic site and that might be characterized by a distinct reaction mechanism. We previously reported the purification and crystallization of 5β-POR from Digitalis lanata (5Herl V. Fischer G. Muller-Uri F. Kreis W. Phytochemistry. 2006; 67: 225-231Crossref PubMed Scopus (47) Google Scholar, 23Egerer-Sieber C. Herl V. Muller-Uri F. Kreis W. Muller Y.A. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2006; 62: 186-188Crossref PubMed Scopus (8) Google Scholar). Here we present two crystal structures of 5β-POR, namely of 5β-POR, in the presence and absence of the cofactor NADP+. The crystal structures show that although 5β-POR displays the standard SDR fold, the architecture of the active site is unprecedented. Protein Production and Purification—To produce recombinant 5β-POR from D. lanata, we slightly modified previously published protocols (5Herl V. Fischer G. Muller-Uri F. Kreis W. Phytochemistry. 2006; 67: 225-231Crossref PubMed Scopus (47) Google Scholar, 23Egerer-Sieber C. Herl V. Muller-Uri F. Kreis W. Muller Y.A. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2006; 62: 186-188Crossref PubMed Scopus (8) Google Scholar). In the pQE-30 expression plasmid (Qiagen, Hilden, Germany), the first 13 residues of the 389-residue-long protein were missing and were replaced by a 6-residue-long N-terminal His tag instead. The best expression levels were obtained in Escherichia coli strain M15[pREP4] at low temperatures. Therefore, two 1-liter LB medium bacteria cultures that were initially grown at 37 °C to an OD of 0.50 were transferred to 12 °C. 5β-POR production was induced at an OD of 0.7 upon addition of 0.3 mm isopropyl 1-thio-β-d-galactopyranoside, and the bacteria cultures were incubated for an additional 48 h. The cells were harvested by centrifugation, and the pellet was dissolved in 5 ml of lysis buffer (NaH2PO4, 300 mm NaCl, 10 mm imidazole, 1 mm (2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, pH 8.0), and the solution was sonicated. Following centrifugation, the filtrated supernatant was loaded onto a 1-ml nickel-Sepharose HP affinity column (GE Healthcare). The protein was eluted with a 20–500 mm imidazole gradient prepared with the lysis buffer described above. 5β-POR was further purified by an additional gel filtration step using a Superdex 75 HiLoad 16/60 column (GE Healthcare) with a 20 mm Tris/HCl, 150 mm NaCl, pH 8.0 buffer. The protein eluted in two separate peaks. Although the first peak contained high molecular weight disulfide cross-linked oligomers that could be visualized in a nonreducing electrophoretic gel and that failed to crystallize, the fractions covering the low molecular weight peak were pooled and concentrated to a final concentration of 21 mg/ml for subsequent crystallization. During the last step, the buffer components were diluted to 6 mm Tris/HCl and 45 mm NaCl, pH 8.0. The overall protein yield was about 1.5 mg of pure protein from 2 liters of bacterial cell culture. Site-directed Mutagenesis and Analysis of Variant 5β-POR—Selective mutants of 5β-POR were constructed by PCR using the Phusion™ site-directed mutagenesis kit (Finnzymes, Finland) with the following primer pairs (substituted amino acids are underlined): Ala-179dir (5′-TGAAGTACATGAACTTTGCCTATGATTTAGAG-3′) and Ala-179rev (5′-ACCTGGGCAAATCCTCAGTGT-3′) for the Y179A mutant; sense Phe-179dir (5′-TGAAGTACATGAACTTTTTCTATGATTTAGAGG-5′) and Ala-179rev for the Y179F mutant. All primers were purchased in a reverse phase-HPLC-purified quality from Eurogentec S.A. (Belgium). The pQE-30 expression plasmid containing wild-type 5β-POR was used as a template (5Herl V. Fischer G. Muller-Uri F. Kreis W. Phytochemistry. 2006; 67: 225-231Crossref PubMed Scopus (47) Google Scholar). PCR was performed in a Personal Cycler 20 (Biometra GmbH, Göttingen, Germany) as follows: 30 s at 98 °C (denaturation), 25 cycles of 10 s at 98 °C (denaturation), 30 s at 68 °C (annealing), and 90 s at 72 °C (extension), finally 5 min at 72 °C (extension). Mutated genes were sequenced 4–6 times (MWG AG, Martinsried, Germany), and the proteins were produced in E. coli (strain M15[pREP4]) as described above for the wild-type protein. To analyze 5β-POR activity, the method described by Stuhlemmer and Kreis (24Stuhlemmer U. Kreis W. Plant Physiol. Biochem. 1996; 34: 85-91Google Scholar) was used and slightly modified (5Herl V. Fischer G. Muller-Uri F. Kreis W. Phytochemistry. 2006; 67: 225-231Crossref PubMed Scopus (47) Google Scholar). The assay contained the following in a final volume of 1000 μl: 945 μl of purified protein fraction (0.2 mg/ml), 6.4 mm NADP+, 32.1 mm glucose 6-phosphate, 42 nanokatals of glucose-6-phosphate dehydrogenase, and 0.3 mm progesterone as substrate. Heat-inactivated (10 min, 100 °C) samples served as controls. The mixtures were kept in 2-ml Eppendorf tubes and incubated at 30 °C and 550 rpm for 2 h prior to extraction, using 1000 μl of dichloromethane. Y179F and Y179A mutants were incubated under standard conditions and prolonged conditions (4 h). The organic phase was evaporated and the pellet dissolved in 50 μl of methanol for subsequent HPLC and TLC analysis. Enzyme activity was calculated using the HPLC method published previously (5Herl V. Fischer G. Muller-Uri F. Kreis W. Phytochemistry. 2006; 67: 225-231Crossref PubMed Scopus (47) Google Scholar). The detection limit was shown to be 80 ng of pregnane-3,20-dione. In addition, the TLC system described by Herl et al. (5Herl V. Fischer G. Muller-Uri F. Kreis W. Phytochemistry. 2006; 67: 225-231Crossref PubMed Scopus (47) Google Scholar), which is about 10 times more sensitive than the HPLC method, was used to check enzyme activity qualitatively. Crystallization and Data Collection—Crystals of 5β-POR with no cofactor bound were grown using the hanging drop method as described earlier (23Egerer-Sieber C. Herl V. Muller-Uri F. Kreis W. Muller Y.A. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2006; 62: 186-188Crossref PubMed Scopus (8) Google Scholar). 1 μl of protein solution was mixed with 1 μl of reservoir solution (15% polyethylene glycol 4000, 0.1 m ammonium acetate, 0.1 m sodium citrate, pH 5.6), and the droplet was suspended over 700 ml of reservoir solution. After 2 days the octahedral crystals with lengths of about 250 μm could be isolated from droplets that were covered by a dense protein skin. The crystals were soaked for 5 min in a cryoprotection solution prepared from 80% (v/v) reservoir solution and 20% (v/v) ethylene glycol prior to being shock-frozen in liquid nitrogen. The binary complex between 5β-POR and NADP+ was prepared by mixing 60 μl of protein solution with an 8-fold molar excess of NADP+. In an attempt to produce the ternary complex consisting of protein, substrate, and cofactor, we incubated the protein solution in addition with 1.8 mg of solid progesterone for 48 h. However, we later did not find any evidence for the presence of progesterone in the structure of the binary complex. After removal of insoluble progesterone by centrifugation, crystals of 5β-POR in complex with NADP+ were grown using the containerless batch method (25Chayen N.E. Protein Eng. 1996; 9: 927-929Crossref PubMed Scopus (59) Google Scholar). 300 μl of high density fluorinated silicon oil (FS-1265 Fluid 10000 CST, Dow Corning, Wiesbaden, Germany) were transferred into a well of a cell culture plate and overlaid with 500 μl of regular silicon oil (silicon oil M 5, Roth, Karlsruhe, Germany). At the interface between the two liquids, a droplet was deposited that was obtained by mixing 0.4 μl of H2O, 2.2 μl of the binary 5β-POR complex solution (see above), and 1.8 μl of a crystallization solution consisting of 22.9% 2-methyl-2,4-petanediol, 3.5% polyethylene glycol 8000, 0.05 m sodium acetate, 0.02 m CaCl2, pH 5.8. Crystals appeared within 48 h and were shock-frozen after being soaked for 5 min in a cryoprotection mixture consisting of the crystallization solution (80%, v/v) supplemented with ethylene glycol (20%, v/v). Highly redundant diffraction data sets of 5β-POR alone and in complex with the cofactor were collected at BESSY synchrotron in Berlin covering a total oscillation range of 180°. Data indexing and processing were accomplished with program XDS (26Kabsch W. J. Appl. Crystallogr. 1988; 21: 916-924Crossref Scopus (1683) Google Scholar). Diffraction data statistics are summarized in Table 1. Both crystals belonged to the tetragonal space group P43212 with cell axes almost identical to those previously observed for the selenomethionine-derivatized crystals of 5β-POR (23Egerer-Sieber C. Herl V. Muller-Uri F. Kreis W. Muller Y.A. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2006; 62: 186-188Crossref PubMed Scopus (8) Google Scholar).TABLE 1Crystallographic data and refinement statistics5β-POR-NADP+ complex5β-PORData collection statistics Unit cell dimensions (Å)a = b = 78.59 c = 178.10a = b = 78.66 c = 180.99 Space groupP43212P43212 Resolution rangeaValues in parentheses refer to the highest resolution shell40–2.3 (2.4–2.3)40–2.4 (2.56–2.4) Wavelength (Å)0.95370.9537 Matthews coefficient (Å3 Da–1)/solvent content (%)3.3/63.23.4/63.8 No. of observationsaValues in parentheses refer to the highest resolution shell367,230 (43,871)438,537 (58,111) No. of unique reflectionsaValues in parentheses refer to the highest resolution shell25,629 (3008)23,196 (3988) RedundancyaValues in parentheses refer to the highest resolution shell14.3 (14.6)18.9 (14.6) Completeness (%)aValues in parentheses refer to the highest resolution shell99.9 (100.0)99.9 (100.0) Mean I/σIaValues in parentheses refer to the highest resolution shell26.0 (4.6)33.7 (5.6) Rsym (%)aValues in parentheses refer to the highest resolution shell7.6 (70.9)5.7 (58.8) Rmeas (%)aValues in parentheses refer to the highest resolution shell7.8 (73.5)5.8 (60.9) Wilson B-factor (Å2)49.562.6Refinement statistics Molecules/AU11 Total no. of atoms30962927 No. of solvent molecules157139 No. of ions1 × Na+, 1 × Cl–1 × Na+ Rvalue17.120.5 Rfree21.225.1 Ramachandran plot (%)bData were calculated with program PROCHECK (most favored regions/allowed/generously allowed/disallowed)93.7/5.7/0.6/0.092.8/5.9/1.3/0.0 Average B-factors (Å2) All atoms (Å2)55.869.5 Protein atoms (Å2)56.370.0 NADP+ atoms (Å2)45.1 Solvent atoms (Å2)48.958.3 R.m.s.deviation bond lengths (Å)0.0120.014 R.m.s. deviation bond angles (°)1.31.4a Values in parentheses refer to the highest resolution shellb Data were calculated with program PROCHECK Open table in a new tab Structure Determination and Refinement—The phases derived from a three-wavelength MAD experiment of selenomethionine-derivatized 5β-POR were of such quality that the chain could be traced entirely de novo (for examples of the experimentally phased 2.7 Å electron density see Ref. 23Egerer-Sieber C. Herl V. Muller-Uri F. Kreis W. Muller Y.A. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2006; 62: 186-188Crossref PubMed Scopus (8) Google Scholar). Because the crystals of 5β-POR alone and in complex with the cofactor NADP+ were isomorphous to the selenomethionine-derivatized 5β-POR, the model built into the MAD-derived density map could be readily transferred to the new diffraction data and completed for missing residues, side chain placements, the bound cofactor, and solvent molecules. An identical set of reflections was used in all structures to validate model building and refinement using Rfree (27Brunger A.T. Acta Crystallogr. Sect. D Biol. Crystallogr. 1993; 49: 24-36Crossref PubMed Google Scholar). Model building was performed using program COOT, and σA-weighted electron density maps were used for guidance (28Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23387) Google Scholar, 29Read R.J. Acta Crystallogr. Sect. A. 1986; 42: 140-149Crossref Scopus (2036) Google Scholar). The model was refined with program REFMAC (30Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13870) Google Scholar), and during the final stages of the refinement, rigid body anisotropic B-factors were introduced using TLS refinement (31Winn M.D. Isupov M.N. Murshudov G.N. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 122-133Crossref PubMed Scopus (1651) Google Scholar). Two different rigid body groups were outlined, namely the double Rossman fold that consists of 5β-POR residues 25–208, 250–279, and 353–368, and the substrate-binding helical domain formed by residues 209–249, 280–352, and 369–389. In both structures a number of solvent-exposed side chains, namely six in the complex structure and 10 in the structure of 5β-POR alone, lacked any electron density and were therefore modeled with atom occupancies of zero. Model building was halted after the refinement of 5β-POR alone and of 5β-POR in complex with NADP+ converged to a final crystallographic Rfactor of 20.2 and 17%, respectively (Table 1). Residual density at position 298 prompted us to resequence the expression construct, thereby confirming that the amino acid at this position was glutamic acid and not glycine (5Herl V. Fischer G. Muller-Uri F. Kreis W. Phytochemistry. 2006; 67: 225-231Crossref PubMed Scopus (47) Google Scholar). A glutamic residue at this position is also present in other 5β-POR orthologs (data not shown). At two positions we observed strong electron density in the cofactor-bound 5β-POR structure that could not be explained by water molecules. One of these could be satisfactorily modeled by a sodium ion and the other as a chloride ion that interacts with Tyr-179 in the enzyme active site. Molecular Modeling of the Ternary Protein Cofactor Substrate Complex—Because any attempts to produce crystals of 5β-POR in complex with either progesterone, cortisol, or 4-androstene-3,17-dione remained unsuccessful regardless whether the cofactor NADP+ was present or not, we computationally docked the substrate progesterone into the binding site. For this purpose, the protein design algorithms of the in-house program MUMBO were supplemented with a flexible ligand-handling routine to identify the energetically most favorable interaction between progesterone and the protein (32Stiebritz M.T. Muller Y.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 2006; 62: 648-658Crossref PubMed Scopus (17) Google Scholar). In a first step a backbone-dependent rotamer library was used to build multiple side chain conformations into the model (33Dunbrack Jr., R.L. Cohen F.E. Protein Sci. 1997; 6: 1661-1681Crossref PubMed Scopus (669) Google Scholar). In addition, up to several thousand random ligand orientations and positions were generated, starting from the coordinates of a manually placed ligand. In the next step, the energetically most favorable combination of side chain conformations and ligand position were identified using either the dead end elimination or the Metropolis Monte Carlo search algorithm in combination with an empirical force field. The force field included in addition to standard terms also a solvation free energy estimate and an empirical H-bond energy term (32Stiebritz M.T. Muller Y.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 2006; 62: 648-658Crossref PubMed Scopus (17) Google Scholar, 34Lazaridis T. Karplus M. Proteins. 1999; 35: 133-152Crossref PubMed Scopus (1117) Google Scholar, 35Kortemme T. Morozov A.V. Baker D. J. Mol. Biol. 2003; 326: 1239-1259Crossref PubMed Scopus (430) Google Scholar). The docking procedure resembles that described by Leach (36Leach A.R. J. Mol. Biol. 1994; 235: 345-356Crossref PubMed Scopus (304) Google Scholar). A more detailed description of the method and validation calculations are provided in supplemental Figs. 1 and 2 and supplemental Tables I and II). The Structure of 5β-POR—The structure of 5β-POR from D. lanata was solved in complex with the cofactor NADP+ at a resolution of 2.3 Å (Rfactor = 17.0%, Rfree = 21.3%) and with no cofactor bound at 2.4 Å resolution (Rfactor = 20.2%, Rfree = 24.9%) (Table 1). In both structures, no electron density was visible for the N-terminal residues 14–25. At present it cannot be decided whether the entire 25-residue-long N terminus in 5β-POR is highly flexible or whether these residues lack any ordered structure because of the deletion of wild-type residues 1–13 in the expression plasmid. Although in the cofactor-bound structure the main chain could be traced contiguously from residues 26–389, in the cofactor-free structure, the segments 68–72 and 155–158 could not be built. All residues in both structures lie within allowed regions of the Ramachandran plot with 93.7 and 92.8% of the residues in the mostly favored regions (37Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). 5β-POR exhibits a typical SDR fold (10Kallberg Y. Oppermann U. Jornvall H. Persson B. Eur. J. Biochem. 2002; 269: 4409-4417Crossref PubMed Scopus (340) Google Scholar), and the N terminus contains the standard dinucleotide-binding double Rossman fold (9Rossman M.G. Liljas A. Branden C.-I. Banaszak L.J. Boyer P.D. The Enzymes. 3rd Ed. Academic Press, New York1975: 61-102Google Scholar, 38Rossman M.G. Argos P. J. Mol. Biol. 1976; 105: 75-95Crossref PubMed Scopus (253) Google Scholar) (Fig. 1). A short insertion that is present in the loop connecting strands βE and βF as well as two long insertions that occur between the sixth and the seventh β-strand (strands βF and βG) and following strand βG are typical for the extended members of the SDR family (18Persson B. Kallberg Y. Oppermann U. Jornvall H. Chem. Biol. Interact. 2003; 143–144: 271-278Crossref PubMed Scopus (175) Google Scholar). The latter two insertions are almost exclusively formed by α-helices and accommodate close to 130 residues of the total 389 residues of 5β-POR. Because of the size of these insertions and because the segments that connect them to the central double Rossman fold shape the steroid-binding pocket, it see
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