Unusual Charge Stabilization of NADP+ in 17beta -Hydroxysteroid Dehydrogenase*

Catherine Mazza, Rock BretonDagger , Dominique Housset§, and Juan Carlos Fontecilla-Camps§

From the Laboratoire de Cristallographie et Cristallogenèse des Protéines, Institut de Biologie Structurale J.-P. Ebel, CEA-CNRS, 41, avenue des Martyrs, F-38027 Grenoble cedex, France

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Type 1 17beta -hydroxysteroid dehydrogenase (17beta -HSD1), a member of the short chain dehydrogenase reductase (SDR) family, is responsible for the synthesis of 17beta -estradiol, the biologically active estrogen involved in the genesis and development of human breast cancers. Here, we report the crystal structures of the H221L 17beta -HSD1 mutant complexed to NADP+ and estradiol and the H221L mutant/NAD+ and a H221Q mutant/estradiol complexes. These structures provide a complete picture of the NADP+-enzyme interactions involving the flexible 191-199 loop (well ordered in the H221L mutant) and suggest that the hydrophobic residues Phe192-Met193 could facilitate hydride transfer. 17beta -HSD1 appears to be unique among the members of the SDR protein family in that one of the two basic residues involved in the charge compensation of the 2'-phosphate does not belong to the Rossmann-fold motif. The remarkable stabilization of the NADP+ 2'-phosphate by the enzyme also clearly establishes its preference for this cofactor relative to NAD+. Analysis of the catalytic properties of, and estradiol binding to, the two mutants suggests that the His221-steroid O3 hydrogen bond plays an important role in substrate specificity.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The 17beta -estradiol (E2) is known to promote the genesis and development of human breast cancers (1, 2). Its presence in tumor cells comes from in situ synthesis (3), and its concentration is significantly increased in malignant breast tissues (4-6). Type 1 17beta -hydroxysteroid dehydrogenase (17beta -HSD1)1 catalyzes the reversible transformation of estrone (E1) into the biologically active estradiol (E2) (7). Thus, preventing the formation of E2 by a specific inhibition of 17beta -HSD1 activity should be of paramount importance for cancer therapy.

The 17beta -HSD1 (Mr = 34,900, 327 residues) is active as a homodimer (8) and requires a dinucleotide cofactor (either NADP+/NADPH or NAD+/NADH) to transform estrogens. Amino acid sequence alignments revealed that it belongs to the short chain dehydrogenase reductase family (SDR) (9-11). A consensus sequence Tyr-Xaa-Xaa-Xaa-Lys (Tyr155 and Lys159 in 17beta -HSD1) as well as a generally conserved serine residue (Ser142 in 17beta -HSD1) characterize this protein family. A catalytic role for the conserved tyrosine and lysine residues has been suggested by site-directed mutagenesis experiments (12-16), and subsequent crystallographic studies (17-26) have confirmed the importance of the consensus triad. In the 17beta -HSD1·E2·NADP+ complex (25), Tyr155, Ser142, and the E2 O17 atom form a triangular hydrogen-bonding arrangement that may result in easier deprotonation of the putatively reactive tyrosine. Lys159 participates in the NAD(P)+ stabilization by establishing hydrogen bonds with O2' and O3' of the nicotinamide ribose.

Based on amino acid sequence alignments, 17beta -HSD1 was thought to belong to the group of NAD(H)-preferring enzymes (22). However, biochemical studies (7) and the structure of the 17beta -HSD1·E2·NADP+ ternary complex (25) have shown that 17beta -HSD1 is able to bind both NAD(H) and NADP(H). 17beta -HSD1 appears to be unique among the SDR family because it lacks both the aspartic acid residue at position 36 (Leu36 in 17beta -HSD1), characteristic of NAD(H) preferring enzymes, and the basic residue located in the consensus sequence of the dinucleotide binding motif Gly-Xaa-Xaa-Xaa-Gly-Xaa-Gly (which is replaced by Ser12 in 17beta -HSD1). This motif forms an ionic interaction with the ribose 2'-phosphate and is characteristic of NADP(H)-preferring enzymes.

His221, first identified by affinity labeling studies (27-29), was thought to be involved in the specific binding of the steroid. Indeed, the construction of an H221A mutant led to an enzyme displaying a higher Km and lower Vmax relative to the wild type (16). Furthermore, the crystal structure of the enzyme complexed with E2 (25, 26) revealed that His221 is directly involved in the specific binding of the steroid.

Here, we report the construction of H221L and H221Q mutants, their characterization by enzymatic assays, their crystallization, and the determination of the structures of the binary complexes H221Q·E2 and H221L·NAD+ and the ternary complex H221L·NADP+·E2 at 2.7, 3.0, and 2.7 Å resolution, respectively. We show for the first time a well ordered conformation for the 191-199 loop and speculate about its role in cofactor binding and hydride transfer. Moreover, the specificity of the enzyme for estrogens is reassessed.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Spodoptera frugiperda, purified AcNPV DNA (Autographa Californica nuclear polyhedrosis virus), and transfer vector pVL1393 were purchased from Invitrogen Corporation; Grace's insect cell culture medium, yeastolate, lactalbumin hydrolysate, fetal bovine serum, and restriction enzymes were from Life Technologies Inc.; NAD+, NADP+, estradiol, and estrone were from Sigma; and Blue-Sepharose CL-6B was from Amersham Pharmacia Biotech.

Cell Culture and Virus-- The TNM-FH medium was prepared from Grace's medium by the addition of 3.3 g/liter yeastolate and 3.3 g/liter lactalbumin hydrolysate. The Sf9 cells were grown as monolayers at 28 °C in TNM-FH medium supplemented with 10% fetal bovine serum. Cells were infected with virus at a multiplicity of infection of 0.1-1 plaque-forming unit to produce virus stocks or at multiplicity of infection >10 for maximal protein expression. Cells were harvested 60 h after infection.

Site-directed Mutagenesis-- The two mutants H221Q and H221L were constructed with one round of polymerase chain reaction made on the pVL/17beta -HSD transfer vector previously constructed for 17beta -HSD1 overexpression in baculovirus (30). These mutations use two primers. The first one is located on the cDNA, upstream of a PstI unique site (5'-GTAGTAGGGACTGTGCGG-3'). The second one introduces the mutation. For H221Q and H221L, it overlaps the codon to be mutated and a NruI unique site (5'-GCCGCCTCGCGAAAGACTTGCTTGCTTTGGGCGAGG-3' and 5'-GCCGCCTCGCGAAAGACTTGCTTGCTGAGGGCGAGG-3', respectively). Amplified fragments and pVL/17beta -HSD1 were digested with NruI and PstI. The mutated fragments were then cloned instead of the nonmutated one. Mutations were checked by dideoxynucleotide sequencing, and mutated transfer vectors were cotransfected in Sf9 cells with the wild-type AcNPV virus following the protocol described by Invitrogen. Recombinant viruses were purified by plating.

Protein Purification and Enzyme Assay-- The purification of mutant proteins was performed as described previously for the wild-type protein (30, 25). The only modification concerned the H221L mutant which was purified at pH 7.0 instead of pH 7.5. The activities of the wild-type and the mutated 17beta -HSD1s were measured as described by Langer and Engel (31) with few modifications. The enzymes were assayed by spectrophotometric measurement of the concentration changes of NADPH at 340 nm. Reactions were run in 50 mM NaHCO3/Na2CO3 buffer, pH 9.2, for the oxidation and 50 mM KH2PO4/K2HPO4 buffer, pH 5.8, for the reduction, both containing 0.5-16 µM steroid (estradiol or estrone, respectively). In both cases, 10 µl of enzyme preparation (from 55 to 90 µg/ml) were added to 500 µl of buffer. Reactions were initiated by the addition of 5 µl of a 10 mM NADP+ or NADPH solution, and the absorbance variations were measured against a blank containing all components except steroid and coenzyme. Controls containing all components except steroid were also run. Reactions were followed from 30 s to 3 min after addition of NADP+ on a Shimadzu UV-160A spectrophotometer. For NADP+ reduction measurements, the same protocol was used except that the estradiol concentration was fixed to 25 µM while NADP+ concentrations varied from 0.05 up to 5 mM. Velocities were calculated from the slopes of the zero order portion of the kinetics obtained and were corrected for the control absorbance. The enzymatic activity was calculated as described previously (8) and defined in units per milliliter (taking a micromole per min as a unit). Lineweaver-Burk plots were used to determine the Km and the Vmax values. Protein concentrations of enzyme preparations were measured using a micro BCA protein reagent kit from Pierce. The catalytic activity parameters are presented in Tables IV and V.

Crystallization-- All recombinant mutant enzymes were concentrated to 5 mg/ml, in a buffer containing 40 mM Tris, pH 7.5, 1 mM EDTA, 0.2 mM DTT, 20% glycerol, and 2 mM beta -octylglucoside and crystallized at room temperature. The H221Q mutant was crystallized in conditions similar to those of the wild-type enzyme (100 mM HEPES, pH 7.0, 100 mM MgCl2, 0.5 mM E2, 2-4% propanediol, 30% polyethylene glycol 4000) (25, 32); crystals appeared after 6 days. The H221L mutant was crystallized in 100 mM sodium phosphate buffer, pH 6.0-6.3, 1 mM NAD(P)+, 100 mM NaCl and 2.2-4.4 mM decyl-beta -D-maltoside or 9-18 mM octyl-beta -D-thioglucopyranoside and 2-2.4 M ammonium sulfate as precipitant. Space groups and unit cell dimensions of these crystals are summarized in Table I.

The H221L crystals used were incubated (from a few minutes up to one night) in a buffer containing 100 mM HEPES, pH 6.5, 1 mM NADP+, 100 mM NaCl, 30% PEG 4000 with and without 0.5 mM E2 to obtain both the ternary and the binary complex. Crystals were then flash-cooled under a nitrogen gas stream at -150 °C, transferred into liquid propane at -160 °C, and conserved in solid propane at -196 °C until data collection was carried out at a synchrotron radiation source.

Data Collection and Processing-- A data set of the H221Q mutant was collected at room temperature on the in-house Siemens/Xentronics area detector X1000 system, mounted on a Rigaku RU200 rotating-anode-x-ray generator. Two data sets of the H221L mutant were collected at low temperature (-150 °C) on the D2AM French-Collaborative Research Group synchrotron beam line at the European Synchrotron Radiation Facility (ESRF, Grenoble), using an XRII-CCD detector (33). Data were processed with XENGEN (34) for the former and with a modified version of XDS (35, 36) for the others. Data collection statistics are summarized in Table I.

                              
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Table I
Data collection statistics
Cell dimensions of the crystals of wild-type enzyme complexed with NADP+ and estradiol (25): a = 122.8 Å, b = 43.8 Å, c = 60.5 Å, alpha  = gamma  = 90°, beta  = 99.5°.

R<SUB><UP>sym</UP></SUB>=<LIM><OP>∑</OP><LL><UP><B>h</B></UP></LL></LIM> <LIM><OP>∑</OP><LL><UP>i</UP></LL></LIM>‖I<SUB><UP>i<B>h</B></UP></SUB>−⟨I⟩<SUB><UP><B>h</B></UP></SUB>‖/<FENCE><LIM><OP>∑</OP><LL><UP><B>h</B></UP></LL></LIM> <LIM><OP>∑</OP><LL><UP>i</UP></LL></LIM> ⟨I⟩<SUB><UP><B>h</B></UP></SUB></FENCE>

Structure Determination-- For the H221Q mutant, a rigid body refinement with the model of the wild-type 17beta -HSD1 was sufficient to place it correctly in the unit cell. The H221L mutant structure was solved by molecular replacement methods with the wild-type 17beta -HSD1 as a search model using AMoRe (37). Four molecules per asymmetric unit were found. The mutants structures were refined with X-PLOR (Version 3.1) (38) and REFMAC (CCP4 Suite of Programs) (39), and model corrections were made with O (40). The refinement statistics are presented in Table II. The coordinates have been deposited with the Protein Data Bank (codes: 1FDU for the H221L·E2·NADP+ complex, 1FDV, for the H221L·NAD+ complex, and 1FDW for the H221Q·E2 complex).

                              
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Table II
Refinement statistics

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Two different crystalline complexes were obtained with the H221L mutant: the first one by co-crystallizing the protein with NADP+ and diffusing E2 in the crystal, and the second one by crystallizing the protein in the presence of NAD+. Both complexes crystallize in the monoclinic P21 space group (Table I) with four molecules in the asymmetric unit corresponding to two biologically active dimers (Fig. 1), here named mA/mB and mC/mD, respectively.


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Fig. 1.   The H221L·E2·NADP+ complex. The non-crystallographic 2-fold axis is shown between the two monomers. The two Rossmann-fold motifs (beta A-alpha B-beta B-alpha C-beta C-alpha D-beta D) are depicted in light pink and light blue. Lys195, Arg37, and Ser11 (yellow) interact with the NADP+ 2'-phosphate. Arg37 and Ser11 belong to Rossmann-fold loops while Lys195 is located in a variable domain. (This figure was generated with Molscript (48) and Render (49, 50).)

In the H221L·NADP+·E2 ternary complex, the electron density is very well defined all along the polypeptide chain, including the 191-199 loop that was disordered in the wild-type structure (Fig. 2). The observed conformation for this loop is completely different from that proposed by Ghosh et al. (24) but the 190-192 segment conformation is similar to that of residues 184-186 in mouse lung carbonyl reductase (MLCR) (22) (Fig. 3). The electron density map is also very well defined for the steroid and cofactor (Fig. 4). As a result, NADP+ and estradiol have been modeled with full occupancies in the four subunits.


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Fig. 2.   Stereoscopic view of the (2Fo-Fc) electron density map around the 189-200 loop of the monomer mC of the H221L·E2·NADP+ complex. The map, computed with 2.7 Å resolution data, is contoured at the 1 sigma  level.


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Fig. 3.   Superposition of the 187-209 loop of the wild-type 17beta -HSD1 model (1bhs (24)) (medium gray) and the monomer mC of the H221L·E2·NAD+ complex (black) with the 182-203 loop of the MLCR model (1cyd (22)) (light gray). Phe192 of the H221L mutant and Met186 of MLCR superpose very well and make extensive hydrophobic contacts with the nicotinamide ring. Lys195 of the H221L model interacts with the 2'-phosphate while no equivalent residue is found for MLCR.


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Fig. 4.   Stereoscopic view of the (2Fo-Fc) electron density map around the NADP+, the residues Ser11 and Arg37 and the 190-196 loop of the monomer mC of the H221L·E2·NADP+ complex. The map, computed with 2.7 Å resolution data, is contoured at the 1 sigma  level.

In the H221L·NAD+ binary complex, the electron density for NAD+ and the 191-200 loop is well defined for monomers mA and mC but discontinuous for monomers mD and mB. Consequently, the 191-199 loop was not included in the models of these two monomers. Since this site is known to have Michaelian kinetics, we do not understand the observed difference in NAD+ occupancy. It may be due to subtle packing effects difficult to characterize at this resolution.

Both NADP+ and NAD+ bind in the same extended conformation already observed for the wild-type enzyme: the cofactor points toward the active site of the enzyme with the nicotinamide ring in the syn conformation and the adenine in the anti conformation (25).

The structure of the H221Q·E2 binary complex displays two conformations for Gln221. In one of these, the amide group of the Gln221 forms a hydrogen bond with the steroid O17 atom; whereas in the other, Gln221 is oriented toward the solvent. The double conformation of the Gln221 may be a consequence of the partial occupation of the steroid-binding site, as it was already suggested in the case of the wild-type enzyme (25). No electron density was found for the cofactor.

Having a complete model of the cofactor binding site allows for a full description of the NAD(P)+/protein interactions. Some of these interactions are common to NADP+ and NAD+, and most of them were present in the wild-type ternary complex (25). However, due to the disorder in the neighborhood of the NADP+ binding site in the latter structure, two major interactions were not observed: the extensive hydrophobic contact between the nicotinamide moiety and the Phe192 side chain, and the charge compensation of the dinucleotide 2'-phosphate through salt bridges with Arg37 and Lys195 side chains (Figs. 4 and 5). In addition, the 2'-phosphate is further stabilized by a hydrogen bond with Ser11 Ogamma . In the NAD+·H221L complex, the binding pocket of the ribose 2'-phosphate of monomers mA and mC is occupied by a sulfate ion, presumably coming from the crystallization solution. This ion is bound to the protein·NAD+ complex through a hydrogen bond network involving 2'-OH adenine ribose, Lys195, Arg37, Ser11, and Thr41 side chains. This phenomenon has already been observed in glutathione reductase where an inorganic phosphate ion substitutes for the missing 2'-phosphate group when NAD+ is bound (41). In both NAD+ and NADP+ complexes, the NH3+ group of Lys195 interacts with the O1A of the pyrophosphate.


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Fig. 5.   The NADP+ environment. The 191-199 loop is represented in black, and NADP+ and estradiol are in medium gray. The 2'-phosphate group is stabilized by three interactions (dashed lines): two salt bridges with Arg37 and Lys195 and a hydrogen bond with Ser11. Phe192 protects the nicotinamide from solvent by an extensive hydrophobic contact. The catalytic site formed by Ser142, Tyr155, the steroid O17 atom, and nicotinamide are also represented. Dashed lines represent the triangular hydrogen bond arrangement between Ser142, Tyr155, and O17.

The 191-199 loop, located between the beta F sheet and the alpha G helix, seems to be predominantly stabilized by its interactions with the dinucleotide, particularly by the salt bridge between Lys195 and the 2'-phosphate. In the H221L·NAD+ complex, where a sulfate ion replaces the 2'-phosphate, the electron density corresponding to the 191-199 loop is less well defined even though the NAD+ site appears to be fully occupied. This implies that although the sulfate ion establishes a series of interactions with both the protein and the cofactor, these are less efficient in stabilizing the 191-199 loop than those formed by the covalently bound 2'-phosphate. Once stabilized, the 191-199 loop appears to protect the coenzyme from solvent as the NADP+ accessible surface (42) is reduced from 122 Å2 when the loop is removed to 38 Å2 when it is well ordered.

The E2 molecule and the residues forming the steroid binding site are well superposed in the two wild-type structures (Table III). On the other hand, the rms differences values resulting from the superposition of each mutant onto the wild-type models show a significant deviation for estradiol, relative to the residues involved in the hydrophobic site (Fig. 6). In this respect, the loss of a hydrogen bond between residue 221 and estradiol may be responsible for the increased steroid mobility observed in the H221L mutant. There is also a significant difference in substrate position between two non-equivalent monomers such as mC and mD. The slight substrate reorientation observed in the H221Q mutant is likely to be due to a shift of the hydrogen bond between E2-O3 and Gln221-Nepsilon 1.

                              
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Table III
Estradiol positional differences
Superpositions were made with the ALIGN program (52) on the substrate binding site residues (Ser142, Val143, Met147, Leu149, Try155, Pro187, Try218, Val225, Phe226, Phe259, and Met279). Monomer mC of the H221L · E2 · NADP+ complex was used for the superposition with the wild-type enzyme. 1fds and 1fdt are Protein Data Bank accession codes for 17beta -HSD1 · E2 and 17beta -HSD1 · E2 · NADP+ complexes, respectively.


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Fig. 6.   Stereoscopic view of the steroid binding site of wild-type (1fdt model in yellow, and 1fds model in orange), H221L (monomers mC in blue, mD in light blue), and H221Q (pink). Phe226 of the 1fdt model and Gln221 of the H221Q mutant are shown with their two modeled conformations. The steroid environment is very well conserved, especially Phe155 and Ser142 which are the residues involved in the catalytic reaction. Significant deviations are observed for the steroid, especially for its C and D rings.

The catalytic efficiency is strongly affected by mutations at the His221 position (Table IV). Reduction is decreased 10-fold for the H221L mutant and 4-fold for the H221Q mutant (18-fold and 6-fold, respectively, for the oxidation). This may be partially explained by a 4.4-fold increase in the reduction reaction Km for H221L and a respective 2.3-fold increase for the H221Q mutant (4-fold and 3.4-fold, respectively, for the oxidation reaction). As the histidine-to-glutamine mutation preserves the hydrogen bond with C3-OH, this interaction seems to be essential for the catalytic activity. As expected from the structure, the H221L mutation has a limited effect on the cofactor binding site architecture, the Km value for NADP+ reduction being close to that of the wild-type enzyme (Table V).

                              
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Table IV
Catalytic activity linked to the steroid binding site

                              
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Table V
NADP+ reduction

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The Role of the 191-199 Loop-- The H221L mutant provides the first image of a well ordered 191-199 loop in a 17beta -HSD1 structure. The conformation of this loop in the H221L·E2·NADP+ complex is very likely to be identical to that of the wild-type enzyme when fully complexed to the cofactor. This is supported, on the one hand, by biochemical results that indicate that the affinity of the H221L mutant enzyme for NADP+ is similar to that of the wild-type protein (Table V) and, on the other, by the fact that the 191-199 loop is located at the protein surface, and it is not involved in potentially constraining crystal packing interactions in either structure. The stabilization of the 191-199 loop, that is disordered in the absence of cofactor, is mediated by the interactions of Lys195 and Phe192 with NADP+. Furthermore, loop residues Phe192 and Met193 shield NADP+ from solvent and contribute to the hydrophobic character of the nicotinamide binding pocket.

Other dehydrogenases also have a similarly flexible loop near their dinucleotide binding site. One example is the mobile loop comprising residues 16-20 in Escherichia coli dihydrofolate reductase which is involved in hydride transfer (43). This loop, which becomes well ordered only in the enzyme·NADP+·folate complex, has been found to shield the nicotinamide moiety from solvent and to participate on the transition-state stabilization (44). Replacement of the Met16-Ala19 stretch by glycine results in a 550-fold decrease in the hydride transfer rate. Another case is lactate dehydrogenase. As in 17beta -HSD1, a loop comprising residues 97-123 is stabilized by interactions with NADH and shields the cofactor from solvent (45). In the SDR family, E. coli 7alpha -HSD (23) exhibits a large conformational change of the 195-210 loop upon substrate binding that also results in shielding of the catalytic site. In the 3alpha ,20beta -HSD structure (19), a small conformational change of the 184-189 loop located close to the active site is also observed. In the MLCR·NADP+ complex (22), the Met186 interaction with the nicotinamide moiety is similar to the one observed between Phe192 and the NADP+ in 17beta -HSD1 (Fig. 3). However, it is not known whether the 184-190 loop has the same conformation in the MLCR apoenzyme.

All the SDR loops described above are located between the beta F sheet and the alpha G helix and are flanked by two proline residues (Pro187 and Pro200 in 17beta -HSD1). As it was previously suggested by Tanaka et al. (23), these two prolines may prevent conformational changes from propagating to the rest of the protein. An amino acid sequence alignment of SDR enzymes reveals that a proline residue located at position 185 (17beta -HSD1 numbering) is highly conserved, except for 17beta -HSD1, where this residue is found at position 187 (Fig. 7). Furthermore, a hydrophobic residue, equivalent to Phe192 of 17beta -HSD1, is found in 38 out of the 46 amino acid sequences analyzed (Fig. 7). These loops, that are stabilized by both substrate or cofactor, may be characteristic of enzymes belonging to the SDR family. In turn, their protection of the nicotinamide moiety and contribution of hydrophobic residues to the active site structure suggest they are involved in catalytic hydride transfer.


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Fig. 7.   Sequence alignment of the known NADP+-preferring SDR enzymes in the 1-50 and 185-200 (17beta -HSD1 numbering) regions. Homology rates above 60% (based on Risler table (51)) are depicted in red. Residues Ser11, Arg37, Phe192, and Lys195 are labeled with a red triangle, Gly9, Gly13, and Gly15 are labeled with a cyan circle, and Pro187 is labeled with a green star. The alignment was obtained with CLUSTALW and corrected with the help of a structural alignment of all the known SDR-structures. Shown are: DHB1_HUMAN, human type 1 17beta -hydroxysteroid dehydrogenase; DHB1_MOUSE, mouse type 1 17beta -hydroxysteroid dehydrogenase; DHB1_RAT, rat type 1 17beta -hydroxysteroid dehydrogenase; AP27_MOUSE, mouse lung carbonyl reductase; DHB3_HUMAN, human type 3 17beta -hydroxysteroid dehydrogenase; BA71_EUBSP, Eubacterium 7alpha -hydroxysteroid dehydrogenase; DHCA_HUMAN, human 15 hydroxyprostaglandin dehydrogenase; DHCA_MOUSE, mouse 15 hydroxyprostaglandin dehydrogenase; DHCA_RABBIT, rabbit 15 hydroxyprostaglandin dehydrogenase; DHCA_RAT, rat 15 hydroxyprostaglandin dehydrogenase; DHG1_BACME, Bacillus megaterium glucose-1-dehydrogenase I; DHG2_BACME, Bacillus megaterium glucose-1-dehydrogenase II; DHG3_BACME, Bacillus megaterium glucose-1-dehydrogenase III; DHG4_BACME, Bacillus megaterium glucose-1-dehydrogenase IV; DHGA_BACME, Bacillus megaterium glucose-1-dehydrogenase-A; DHGB_BACME, Bacillus megaterium glucose-1-dehydrogenase-B; DHG_BACME, Bacillus megaterium glucose-1-dehydrogenase; DHG_BACSU, Bacillus subtillis glucose-1-dehydrogenase; DHI1_HUMAN, human corticosteroid 11beta -dehydrogenase; DHI1_MOUSE, mouse corticosteroid 11beta -dehydrogenase; DHI1_RAT, rat corticosteroid 11beta -dehydrogenase; DHI1_SHEEP, sheep corticosteroid 11beta -dehydrogenase; DLTE_BACSU, Bacillus subtillis DLTE protein; FABG_ARATH, Arabidopsis thaliana 3-oxoacyl-[acyl-carrier protein] reductase; FABG_BRANA, Brassica napus 3-oxoacyl-[acyl-carrier protein] reductase; FABG_CULPA, Cuphea lanceolata 3-oxoacyl-[acyl-carrier protein] reductase; FABG_ECOLI, Escherichia coli 3-oxoacyl-[acyl-carrier protein] reductase; FABG_HAEIN, Hemophilus influenzae 3-oxoacyl-[acyl-carrier protein] reductase; FABG_VIBHA, Vibrio harveyi 3-oxoacyl-[acyl-carrier protein] reductase; GNO_GLUOX, Gluconobacter oxydans gluconate 5-dehydrogenase; HDHA_CLOSO, Clostridium sordellii 7alpha - hydroxysteroid dehydrogenase; PHAB_ACISP, Acinetobacter sp. acetoacyl_CoA reductase; PHAB_PARDE, Paracoccus denitrificans acetoacyl_CoA reductase; PHBB_ALCEU, Alcaligenes eutrophus acetoacyl_CoA reductase; PHBB_CHRVI, Chromatium vinosum acetoacyl_CoA reductase; PHBB_RHIME, Rhizobium meliloti acetoacyl_CoA reductase; PHBB_ZOORA, Zoogloea ramigera acetoacyl_CoA reductase; PTR1_LEIMA, Leishmania major pteridine 1 reductase; PTR1_LEITA, Leishmania tarentolae pteridine 1 reductase; ROH2_RAT, rat type 2 retinol dehydrogenase; ROH3_RAT, rat type 3 retinol dehydrogenase; TRN1_DATST, Datura stramonium tropinone reductase-I; TRN2_DATST, Datura stramonium tropinone reductase-II; TRN2_HYONI, Hyoscyamus niger tropinone reductase-II; TRNH_DATST, Datura stramonium tropinone reductase; and YCIK_ECOLI, Escherichia coli hypothetical oxidoreductase in btur-sohb intergenic region.

The Coenzyme Specificity-- As indicated by the different x-ray complex structures, and by previous biochemical results (7), 17beta -HSD1 is able to bind both NADP+ and NAD+ cofactors. The 17beta -HSD1·NADP+ complex has been shown to be stabilized through hydrogen bonds established between the dinucleotide 2'-phosphate moiety and the main chain NHs of residues Cys10, Ser11, and Arg37 (25). In most known structures of NADP+-preferring enzymes, the two negative charges of the 2'-phosphate group are compensated by one or two positively charged residues. Due to disorder of the 191-199 loop, these interactions were not observed in our NADP+/wild-type 17beta -HSD1 complex structure.

Now, the H221L mutant structure with its well defined 191-199 loop, allows us to definitely classify 17beta -HSD1 among the NADP+-preferring enzymes. Three residues, Ser11, Arg37, and Lys195, interact with the dinucleotide 2'-phosphate group of the cofactor (Fig. 5). Arg37 forms a salt bridge that compensates one of the two negative charges of the phosphate. Incidentally, this residue, as Arg39, is also present in the MLCR·NADP+ complex (22). In both structures, it is located in the turn between the beta -sheet B and the alpha -helix C of the beta A-alpha B-beta B-alpha C-beta C-alpha D-beta D motif that forms the Rossmann fold (Figs. 1 and 7). This arginine is conserved in 26 of the 46 proteins known to be NADP+-preferring enzymes (46) (Fig. 7).

A second basic residue, located in the 4th position of the Gly-Xaa-Xaa-Xaa-Gly-Xaa-Gly consensus sequence of the dinucleotide binding motif, often further compensates the 2'-phosphate charge. Indeed, sequence alignment studies reveal that this residue is conserved in 54% of the NADP+-preferring SDR proteins (Fig. 7). In 17beta -HSD1, there is no basic residue in the 4th position. Instead, the 2'-phosphate group interacts with Ser11, which is located in the third position of the consensus sequence. In fact, among the 21 NADP+-preferring enzymes of the SDR family that do not have a basic residue at this position, 15 are found to have either serine or threonine instead (Fig. 7). In 17beta -HSD1, further charge compensation of the 2'-phosphate group is afforded by Lys195. This residue is not conserved in the SDR family of amino acid sequences, and superposition of the known SDR structures shows that it is located in a variable loop. Moreover, in most of the NADP+·enzyme complex structures having a Rossmann-fold binding motif (47), the 2'-phosphate group interacts with one to three residues located in either the beta Aalpha B or the beta Balpha C turn (Fig. 1). Thus 17beta -HSD1 is the first SDR structure for which such an atypical charge compensation is observed. This may be relevant to the understanding of the evolution of the NADP(H) binding motif.

The Role of His221 in Substrate Specificity-- In the wild-type structure, His221 was found to hydrogen bond to the C3-OH of the estradiol moiety. This hydrogen bond is preserved in the H221Q mutant. In the H221L mutant, however, the mutated leucine residue can only participate in the formation of the hydrophobic pocket of the steroid binding site. Consequently, it can be concluded that this hydrogen bond is not essential to substrate binding. However, comparison of all the available x-ray structures shows that, as a function of the immediate environment, estradiol can occupy slightly different positions in its hydrophobic pocket and that the largest differences concern its C and D rings (Fig. 6). The lack of hydrogen bond formation in the H221L mutant reduces the catalytic efficiency by 9 to 18-fold. This can be compared with a 4- to 5-fold reduction in the H221Q mutant (Table IV). Taken together, these results suggest that the hydrogen bond between His221 and the C3-OH group is important for enzyme specificity because it helps in establishing the catalytically relevant steroid/protein interactions of O17, Tyr155 Oeta , and Ser142 Ogamma . These observations may explain the specificity of 17beta -HSD1 for aromatic A ring-containing substrates. With substrates like testosterone or 4-androstenedione, the orientation of the C3-O3 bond relative to the C17-O17 bond is different. Accordingly, we suggest that for these substrates, the interaction of His221 with the O3 atom would place the O17 in a position which is unfavorable for catalysis.

    ACKNOWLEDGEMENTS

We thank J.-L. Ferrer and M. Roth (IBS, Grenoble) for help using the D2AM ESRF beam line.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (codes 1FDW, 1FDV, and 1FDU) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

Dagger Recipient of a Postdoctoral Fellowship from the Medical Research Council (MRC) of Canada and a contract "collaborateur temporaire étrangé" of the Commissariat à l'Energie Atomique, France, and present address: Molecular Endocrinology, CHUL Research Center, 2705 boulevard Laurier, Québec, G1V 4G2, Canada.

§ To whom correspondence should be addressed. E-mail: housset{at}lccp.ibs.fr or juan{at}lccp.ibs.fr.

1 The abbreviations used are: 17beta -HSD1, type 1 17beta -hydroxysteroid dehydrogenase; SDR, short chain dehydrogenase reductase; MLCR, mouse lung carbonyl reductase.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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