Three-Dimensional Structure of Holo 3A,20J3-Hydroxysteroid

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Proc. Nati. Acad. Sci. USA Vol. 88, pp. 10064-10068, November 1991 Biochemistry Three-dimensional structure of holo 3a,20j3-hydroxysteroid dehydrogenase: A member of a short-chain dehydrogenase family (x-ray crystaflography/steroid-metabolizing enzyme/dinucleotide-linked oxldoreductase/sterold-protein interaction/sequence and folding homologies) DEBASHIS GHOSH*t, CHARLES M. WEEKS*, PAWEL GROCHULSKI*t, WILLIAM L. DUAX*, MARY ERMAN*, ROBERT L. RIMSAY§, AND J. C. ORR§ *Medical Foundation of Buffalo, 73 High Street, Buffalo, NY 14203; and Memorial University of Newfoundland, St. John's, Newfoundland, Canada AlB 3V6 Communicated by Herbert A. Hauptman, July 18, 1991 (receivedfor review May 14, 1991)

ABSTRACT The x-ray structure of a short-chain dehy- the substrate binding regions, offers further insight concern- drogenase, the bacterial holo 3a,20/3-hydroxysteroid dehydro- ing the significance of conserved residues and their possible genase (EC 1.1.1.53), is described at 2.6 A resolution. This roles in substrate specificity and overall enzyme function. enzyme is active as a tetramer and crystallizes with four identical subunits in the asymmetric unit. It has the a/( fold characteristic ofthe dinucleotide binding region. The fold ofthe MATERIALS AND METHODS rest of the subunit, the quarternary structure, and the nature The crystals, grown in the presence of 4 mM NADH, belong ofthe cofactor-enzyme interactions are, however, significantly to the space group P43212 having unit cell dimensions a = different from those observed in the long-chain dehydrogena- 106.2 A and c = 203.8 A and contain one full tetramer (106 ses. The architecture of the postulated active site is consistent kDa) in the asymmetric unit (13). Native and HgCl2 and with the observed stereospecificity of the enzyme and the fact KAu(CN)2 derivative data sets were collected on a Mark III that the tetramer is the active form. There is only one cofactor multiwire area detector at the Research Resource in Protein and one substrate-binding site per subunit; the specificity for Crystallography, University of California at San Diego. The both 3a- and 2013-ends of the steroid results from the binding native data set had 91,203 observations for 20,738 unique of the steroid in two orientations near the same cofactor at the reflections to 3.0 A resolution with an Rmerg(I) of0.081. Two same catalytic site. Hg and one Au data sets contained 127,285 and 31,590 (Hg) and 94,437 (Au) observations for 32,843 and 15,909 (Hg) and 3a,20f3-Hydroxysteroid dehydrogenase [3a,20f3-HSD; (20R)- 29,666 (Au) reflections, respectively, including the Friedel 17a,20,21-trihydroxysteroid:NAD+ oxidoreductase, EC mates, to 3.3 A. The Rmerge(I) values were 0.109, 0.076, and 1.1.1.53] from Streptomyces hydrogenans is a nicotinamide 0.080, and the R(F)io values were 0.21, 0.17, and 0.11, adenine dinucleotide [NAD(H)]-linked enzyme involved in respectively (see ref. 14 for definition of terms). In addition, the reversible oxidation of the 3a- and 20f3-hydroxyl groups a 3.3 A data set having 29,039 observations and 10,709 unique of androstane and pregnane derivatives. Recent sequence reflections was also collected on a Hg + Au double derivative determination has revealed that 3a,20/3-HSD is a member of [R(fliso = 0.20]. A set of native data to 2.6 A, having 106,145 the nonmetallo-short-chain alcohol dehydrogenase (ADH) observations for 28,726 unique reflections [R.ee(I) = family (1), which includes 11f,-hydroxysteroid (llP-HSD) 0.091], was collected on film from six crystals at the Cornell (2), 7a-hydroxysteroid (3), and 15-hydroxyprostaglandin de- High Energy Synchrotron Source and processed by using hydrogenases (4) from mammals; glucose (5) and ribitol (6) Rossmann's program at Purdue University. The area detec- dehydrogenases, as well as a putative nodulation factor (7) tor data to 3 A and film data between 3 and 2.6 A were merged from bacteria; and an ADH (8) from insects. Enzymes into a composite native data set. The Hg derivatives each had belonging to this family have -250 amino acid residues, a single major binding site per subunit, whereas the Au similar coenzyme specificity, and partial sequence homol- reagent gave a "multiple low-occupancy site" derivative that ogy. Although more than 40 crystal structures of =15 types was used only to 4.5 A with marginal effect in phasing. The of NAD(H)- and NADP(H)-linked dehydrogenase enzymes double derivative had an additional Au site at a special have been determined at medium-to-high resolution (9), to position. Four Hg-binding positions were determined and our knowledge no x-ray crystallographic study describing the refined by programs HASSP and HEAVY (15), respectively three-dimensional structure of a dehydrogenase belonging to (with the Wilson plot scale factor, the occupancies at the four this short-chain class has been reported. This is only the third major sites were refined to 1.00, 1.02, 1.04, and 0.94). The structure ofan enzyme for which steroids are the substrate to choice of enantiomorph was determined from the anomalous be determined by x-ray diffraction techniques. A low- data. The final phasing powers for the Hg, the Au, and the resolution structure of keto-steroid isomerase (10) and the double derivatives were 2.43, 0.90, and 3.01, respectively, refined structure of cholesterol oxidase (11) have been pub- and the centric R values were 0.59, 0.75, and 0.54, respec- lished. tively. The overall figure-of-merit at 3.3 A was 0.61. To account for the ability of 3a,20f3-HSD to transfer a hydride to either end of a steroid molecule, "one steroid-two Abbreviations: HSD, hydroxysteroid dehydrogenase; MIR, multiple cofactor sites" and "two steroid orientations-one cofactor isomorphous replacement; GAPDH, glyceraldehyde-3-phosphate site" models (12) have been proposed. When analyzed in dehydrogenase; LDH, lactate dehydrogenase; MDH, malate dehy- conjunction with sequence homology studies, the three- drogenase; ADH, alcohol dehydrogenase. dimensional structured especially at the cofactor binding and ITo whom reprint requests should be addressed. *Permanent address: Technical University of Lodz, Institute of Physics, 93-005 Lodz, Poland. The publication costs of this article were defrayed in part by page charge IThe atomic coordinates and structure factors have been deposited payment. This article must therefore be hereby marked "advertisement" in the Protein Data Bank, Chemistry Department, Brookhaven in accordance with 18 U.S.C. §1734 solely to indicate this fact. National Laboratory, Upton, NY 11973 (reference 1 HSD). 10064 Downloaded by guest on September 28, 2021 Biochemistry: Ghosh et al. Proc. Natl. Acad. Sci. USA 88 (1991) 10065 Density averaging based on noncrystallographic 222 sym- A metry within the molecular envelope and solvent flattening outside followed by stepwise phase extension from 3.3 A to 3.0 A were carried out next (the program was kindly provided by E. M. Westbrook, Argonne National Laboratory). A total of21,631 reflections having F > 2o(F) in the resolution range 40.0 A-3.0 A (92% of possible data) were phased. The average phase shift, the map Rj,0~ and the final figure-of-merit were 79.60, 0.299, and 0.81, respectively. Fig. 1 shows the typical density for an a-helix of the symmetry-averaged final multiple isomorphous replacement (MIR) map. The model building was done on an Evans and Sutherland PS390 using FRODO (16). The chain tracing was B done for one subunit; the tetramer was generated by the 55 222-symmetry operation. The MIR model was refined with 45 noncrystallographic symmetry restraint by a version of the program PROLSQ (17) by using the CRAY Y-MP computer at 43 the Pittsburgh Supercomputing Center. The resolution was increased in steps to 2.6 A. The conventional R factor from the last cycle with 7424 protein atoms (255 residues and 1856 34~~~~~~~ atoms per subunit) and 176 NAD atoms is 0.231 for 26,753 (F > 2oF) reflections between 6.0 and 2.6 A resolution (R is l8 0.223 for 5.0-2.6 A). This represents 85% of possible data at 28 6.0-2.60 A (92% at 6.0-2.65 A). The rms deviations from ideality for various geometrical parameters are 0.025 A for the bond distance, 0.061 A for the angle distance, and 3.30 for the bond angle. The average rms deviation ofthe main-chain atoms offour subunits from the 222 symmetry is 0.28 A. The average individual temperature factorfor all atoms is 14.8 A2. No solvent (-50% ofthe cell volume is solvent) has yet been included in the model. The residue Asp-66 in the chemical sequence is tentatively Cys-66 in the present model; a Cys at this point is reconcilable with both the MIR and the calculated maps-the St atom of Cys-66 is at a distance of 2.6 ± 0.3 A from the refined Hg position of the isomorphous derivative. A Ramachandran plot of the refined structure shows that six nonglycine residues per subunit are in the disallowed regions; these belong to three loop areas and the carboxyl terminal, all of which have weak densities and very high temperature fac- FIG. 2. (A) Schematic diagram of the structure of 3a,20,(-HSD and the nomenclature of the secondary elements. (B) Stereographic view ofthe numbering ofresidues represented by their respective CO atoms. (C) Ribbon diagram of a subunit. The NAD(H) molecule is shown in pink and a cortisone molecule modeled in the apparent active site is shown in blue. Also shown in this diagram are some important residues: Met-94, Met-184, and Met-189 (yellow); Arg-16, Asp-37, and Lys-156 (blue); Tyr-152 and His-181 (red); and Thr-12, Ser-91, and Ser-139 (white). The protein is viewed approximately perpendicular to the plane ofthe (3-sheet in B and parallel to the plane of the (-sheet in C. tors. These loop areas are between a-helix aC and (-strand (3C, (3C and aD, and 3F and aG (Fig. 2A). RESULTS AND DISCUSSION The Molecular Conformation of the Subunit. Each of the four identical subunits (referred to as subunits A, B, C, and D) of 3a,20P-HSD has a parallel a/(3 structure with a classic doubly wound ,B-sheet (18). Itforms a single domain structure consisting of a P-sheet formed by seven parallel strands having three parallel a-helices on each side. The nomenclature of the secondary structure elements along with the numbering scheme and a stereoview of the numbering of selected Ca atoms of a subunit is shown in Fig. 2 A and B, FIG. 1. Example of the averaged MIR density map at 3.0 A the B resolution shown with the refined atomic model superimposed: an respectively. Fig. 2C is a ribbon diagram of subunit, amino-terminal segment of helix aF that includes the highly con- with the observed NAD(H) molecule and a modeled corti- served segment Tyr-Gly-Ala-Ser-Lys (residues 152-156). The map sone in the apparent active site. The nucleotide binding was contoured at the 1.0cr level. region is generated by the (3A--aB-3B-aC-,8C fold. The tight Downloaded by guest on September 28, 2021 10066 Biochemistry: Ghosh et al. Proc. Natl. Acad. Sci. USA 88 (1991)

FIG. 3. Stereodiagram ofthe C0 trace of a tetramer of 3a,203-HSD with the molecular axes P, Q, and R along the three noncrystallographic twofold axes. The subunit colors yellow, green, blue, and red are to be identified with subunit names A, B, C, and D, respectively. Four NAD(H) molecules are shown in pink. Also shown in the figure are two modeled cortisone molecules in catalytic clefts of the yellow and green subunits. These clefts are exposed to the top surface of the tetramer. Two other cortisone molecules in clefts exposed to the bottom surface are not shown. turn from P3A to aB, a characteristic of NAD(H) binding ring and C2N of the nicotinamide (IUPAC-IUB nomencla- regions, is facilitated by the presence of several glycine ture; see ref. 21) is 17 A, 2 A longer than that observed in residues in the turn area, similar to many NAD(H)-binding long-chain dehydrogenases (21, 22), but comparable to the folds (9, 19). The a(3 motif is repeated three more times to 17.1 A value observed for NADPH in dihydrofolate reductase complete the helices aD, aE, and aF on the other side and (23). Like glyceraldehyde-3-phosphate dehydrogenase strands (3D, PE, and f3F such that 3D packs against P3A. The (GAPDH) (24), the nicotinamide ring is in the syn confor- carboxyl-terminal segment of67 residues consists ofthe helix mation. This is consistent with the fact that a B-face hydride aG, parallel to and beside aB, a large 32-amino acid loop AG is transferred in 3a,20(8-HSD-catalyzed reactions (25), as in between aG and PG, the final 3-strand, (3G, and a 16-residue GAPDH. At the present level of clarity, most of the confor- carboxyl-terminal loop. Type I and type II (-turns connect mational angles are in the ranges previously observed for most 3-strands and a-helices. The three helices on each side nucleotide-linked oxidoreductases and in the Li-NAD' crys- ofthe sheet pack together and against the sheet, thus forming tal structure (26). the compact core of the subunit. The nicotinamide end of the molecule is buried in a cleft Subunit Association and Quarternary Structure. Tetrameric surrounded by four segments of the protein subunit: the aggregation of holo 3a,20(3-HSD is illustrated in Fig. 3 with 83A-aB turn formed by residues Ile-11 to Arg-16, residues the twofold symmetry axes P, Q, and R labeled convention- Ala-36 to Leu-39 between (3B and aC, residues Ala-88 to ally (20). The blue-green and the red-yellow pairs ofsubunits Ile-90 of(D, and residue Ile-110 of aE (Fig. 5). The carbox- associate along the (3G strands, forming a two-stranded amide group is at a hydrogen bond-forming distance from the antiparallel (-structure in the middle of a 14-stranded sheet, side chain of Asp-37 (average distance of 3.0 A). The Thr-12 a nearly 180'-twisted surface, that stretches from one subunit and Arg-16 side chains have close contacts to atom C4N ofthe to the other. The P-axis is nearly normal to the middle of the nicotinamide ring (Thr-12 OY *- C4N, 3.0 A; Arg-16 NH1 .. sheet and also to the aG helices that pack against each other C4N, 3.6 A). The ribose ring is close to Ile-110. In contrast, about the axis. The loop AG and the carboxyl-terminal loop NAD(H) in long-chain dehydrogenases is translated more from another subunit, as well as two amino termini, also towards the interior, so that the adenine-ribose moiety has associate about this twofold axis. The blue-green/red- the closest interaction with the region of the protein that is yellow interface, defined by the axis Q, is characterized by near the nicotinamide ring in the 3a,20(-HSD structure. The the interaction of the 3D--aE turn (AD'), aE, PE-aF turn (AE'), and aF region with the corresponding twofold related counterpart, giving rise to helix-helix and helix-loop type packings. This is the interface with closest proximity to the cofactors and thus would regulate any cooperative effect among them. Contacts in the blue-red/green-yellow interface are the sparsest and are achieved through an antiparallel association across the R-axis of eight residues in the carboxyl-terminal "arm" and three residues in loop AE'. How- ever, association across the R-axis forms the edge ofthe cleft that presumably binds the steroid and is essential for the formation of the active tetramer. The structural elements responsible for the steroid-binding clefts pack around the molecular center of the tetramer (overall dimensions = 65 A x 62 A x 54 A) exposing two clefts on each broad surface. This puts the NAD(H) binding region, (3A-aB-(B--aC-(3C- aD, on the exterior of the assembly, with cofactor molecules FIG. 4. NAD(H) molecule fitted into an Fob density map with exposed to the solvent. Structural stability is enhanced by the calculated phases, which also included MIR phases for reflections association of 3D-loop AD'-aE segments of the adjacent below 6 A resolution. A (Fobs - Fat) map computed with only the subunits related by the twofold Q-axis. 6-2.6 A resolution data and calculated phases showed broken and weak densities for cofactors. This map was calculated at 3.0 A and The Binding Environment and Conformation of NAD(H). contoured at 1.0of. The best models of the cofactors were obtained The cleft, generated by amino-terminal ends of a-helices B, by fitting this map. The positional parameters of the cofactor atoms C, and D; carboxyl-terminal ends of (3-strands A, B, and C; were not refined. The refinement of the temperature factors of the and midsections of (D and aE, is the cofactor binding site. cofactor atoms resulted in high values for them, an average of about The NAD(H) molecules are in an extended conformation 40 A2. Small differences in conformations of cofactors at the four (Fig. 4). The average distance between C6A of the adenine binding sites are not considered significant at the present resolution. Downloaded by guest on September 28, 2021 Biochemistry: Ghosh et al. Proc. Natl. Acad. Sci. USA 88 (1991) 10067 adenine moiety at the other end ofthe cofactor is sandwiched between Trp-67 and Ile-117. Some strong additional densities in the proximity ofthe phosphate and ribose oxygens suggest the presence of tightly bound solvent molecules in this region. The other charged side chains that approach the cofactor-binding pocket are Asp-109 and Glu-65. The Architecture of the Presumed Steroid-Binding Site. Shown in Fig. 6 is the active-site cleft belonging to the B (green) subunit. The deep cleft, filled with solvent density, is lined by parts of P-strands D, E, and F, loop AE', the flexible loop between PF and aG, and eight carboxyl-terminal residues from one subunit together with part of loop AG from an adjacent subunit. The B-face ofthe nicotinamide ring is open to the cleft. A cortisone molecule (27) has been fit into this site without altering any observed positions of the protein side chains. The loop Thr-12 to Leu-18 forms the top of the cleft. Other residues lining the steroid binding pocket include Ile-90 to Met-94, Ile-137 to Thr-149, the highly conserved sequence from Tyr-152 to Lys-156, Val-180 to Met-189, and Pro-247 to Tyr-251 from the B (green) subunit; Asp-227 from the C (blue) subunit; and Thr-244 from the A (yellow) subunit. Thus, the predicted Lys and Met in the active site (ref. 28 and references therein) are Lys-156 and Met-184 (also possibly Met-189 and/or Met-94; Fig. 2C). However, the His residue FIG. 6. View of the catalytic site. A cortisone molecule, shown claimed to be "affinity alkylated" by haloacetoxysteroids in pink, is modeled in the pocket. Dotted Van der Waals surfaces of cortisone atoms and proximal protein atoms are depicted in blue and and chemically modified by diethyl pyrocarbonate (ref. 28 red, respectively. Atoms from four different subunits are illustrated and references therein) is His-181, which is buried enough in four colors. Some ofthe residues having potential interaction with (Fig. 2C) to be inaccessible without some rearrangements of the steroid are labeled. The nicotinamide end ofthe cofactor, drawn the active-site structure in this holo form. Atoms from the B in purple, is visible at the upper left-hand corner of the figure. and A subunits pack against the D (red) subunit. The active site is thus comprised ofresidues from all subunits, consistent from the pocket, thereby permitting a closer approach of the with the observation (29) that tetrameric assembly is required steroid and the dinucleotide to each other and a direct hydride for activity of the enzyme. transfer through a much shorter distance. Possible hydrogen bond-forming interactions between cor- Sequence and Fodking Homologies. The alignment of the tisone and the enzyme are 11-keto to Tyr-152 OH and to sequence of 3a,203-HSD with five other members of the Ser-139 OH, 3-keto to Asp-227 COOH ofthe adjacent subunit short-chain dehydrogenase family, glucose dehydrogenase, [subunit C (blue) in Fig. 6], and 17-hydroxyl to Thr-185 CO. 11p-HSD, ribital dehydrogenase, nodulation factor, and 15- These distances are all between 2.5 and 3.5 A. However, hydroxyprostaglandin dehydrogenase, and with pig heart judging from the flexible architecture of the cleft and its lactate dehydrogenase (LDH) (30) was performed by using openness to the surface, it is possible that steroids with the program ALIGN (31). The identity score with 3a,20,-HSD various substitutions, particularly at positions 3, 11, 16, 17, ranged from 25% (for 11p-HSD) to 35% (for glucose dehy- and 20, bind differently in this pocket. In Fig. 6, C4N of the drogenase). Nodulation factor had the highest alignment nicotinamide ring is 8.2 A from the 20-carbonyl position of score of 19 standard deviations. LDH, selected as a repre- cortisone. The side chain of Arg-16 lies between the hydride sentative ofthe NAD(H)-binding long-chain dehydrogenases position of the nicotinamide ring and the 20-keto position of for which the three-dimensional structures are available, had cortisone. The Ng atom of Arg-16 is 2.8 A from the 20-keto a very poor alignment score, 1.1 standard deviations. When oxygen of cortisone, and the NH1 of Arg-16 is 3.6 A from LDH and 3a,20(3-HSD are aligned based on the least-squares C4N. Although there are no documented cases of Arg par- fit of their "Rossmann fold" described below, the identity ticipating in a hydride transfer, the possibility exists here that score of LDH with 3a,208-HSD was only 7%. This result Arg-16 possesses a direct or indirect role in this process. It is clearly demonstrates that, despite a highly analogous fold, also likely that the binding of the substrate in this pocket is these two enzymes belong to different classes. Although the accompanied by changes in the conformation of residues long-chain dehydrogenases have been characterized as hav- lining the pocket. The Arg-16 side chain could swing away ing a highly conserved Gly-Xaa-Gly-Xaa-Xaa-Gly (Xaa =

* LEU

OLT 0 ILE FIG. 5. Protein environment ofthe bound NAD(H) molecule in the C subunit. The main- and side- chain atoms of residues relevant to the discussion in the text are shown and labeled. Downloaded by guest on September 28, 2021 10068 Biochemistry: Ghosh et al. Proc. Natl. Acad. Sci. USA 88 (1991) any amino acid) in the turn 3A-aB (19), the distribution ofthe sources cooperative agreement U41 RR04154); Dr. Zdzislaw conserved glycine residues in the 3a,20(3-HSD class of short- Wawrzak and Leszek Rychlewski (sequence alignment computa- chain dehydrogenases has the following pattern (in 3a,20/3- tions); Dr. R. C. Beavis (mass spectrometric molecular weight HSD): determination); Dr. E. M. Westbrook (density averaging program); J. S. Punzi (help in growing the first data-quality crystal); and Dr. 4- PA aB J. F. Griffin for critically reading the manuscript and making many helpful suggestions. This research was funded by National Institutes Gly-(Xaa)6-Gly-(Xaa)2-Xaa-Gly(Ala)-Xaa-Gly-(Xaa)3-Ala of Health grant no. DK26546. A number of other short-chain dehydrogenases, including 1. Marekov, L., Krook, M. & Jornvall, H. (1990) Fed. Eur. Biochem. human 17j3-hydroxysteroid dehydrogenase, show less homol- Soc. 266, 51-54. ogy with 3a,20f3-HSD. These possibly constitute another 2. Agarwal, A. K., Monder, C., Eckstein, B. & White, P. C. (1989) J. class of short-chain dehydrogenases. Biol. Chem. 264, 18939-18943. Comparison of Short-Chain and Long-Chain Dehydrogena- 3. Franklund, C. V., de Prada, P. & Hylemon, P. B. (1990) J. Biol. ses. The three-turn helix aD of 3a,20(-HSD is absent in Chem. 265, 9842-9849. 4. Krook, M., Marekov, L. & Jornvall, H. (1990) Biochemistry 29, dogfish LDH (32), pig heart cytoplasmic malate dehydroge- 738-743. nase (MDH) (33) and lobster GAPDH (34), but present in 5. Makino, Y., Negoro, S., Urabe, I. & Okada, H. (1989) J. Biol. horse liver alcohol ADH (35). The four-turn helix aC, re- Chem. 264, 6381-6385. sponsible for subunit association perpendicular to the Q-axis 6. Morris, H. R., Williams, D. H., Midwinter, G. G. & Hartley, B. S. in LDH and MDH, is one of the two shortest helices in (1974) Biochem. J. 141, 701-713. GAPDH. The strand (3D and the 7. Debelle, F. & Sharma, S. B. (1986) Nucleic Acids Res. 14, 7453- 3a,20,3-HSD, ADH, and 7472. following loop AD' in 3a,20f3-HSD are similar to those in 8. Benyajati, C., Place, A. R., Powers, D. A. & Sofer, W. (1981) Proc. LDH and MDH in overall size and conformation; this loop is Natl. Acad. Sci. USA 78, 2717-2721. almost nonexistent in ADH and GAPDH. The eight-turn 9. Birktoft, J. J. & Banaszak, L. J. (1984) Peptide Prot. Res. 4, 1-44. helix aE is comparable to helices aE and aDE linked by an 10. Westbrook, E. M., Piro, 0. E. & Sigler, P. B. (1984)J. Biol. Chem. intervening kink in LDH and MDH. The loop AE', which 259, 9096-9103. is not 11. Vrielink, A., Lloyd, L. F. & Blow, D. M. (1991) J. Mol. Biol. 219, lines part of the proposed steroid binding pocket, 533-554. present in long-chain dehydrogenases. The six-turn helix of 12. Sweet, F. & Samant, B. R. (1980) Biochemistry 19, 978-986. 3a,20f-HSD, aF, is shorter in LDH and MDH and absent in 13. Ghosh, D., Punzi, J. S. & Duax, W. L. (1986) J. Biol. Chem. 261, ADH and GAPDH. A least-squares fit of the Ca atoms in af3 1306-1308. motifs of the NAD(H) binding fold of 3a,20,3-HSD with those 14. Blundell, T. L. & Johnson, L. N. (1976) Protein Crystallography in these four dehydrogenases demonstrates that the maxi- (Academic, London). mum number of Ca atoms similar to 3a,20j-HSD exists in 15. Terwilliger, T. C. & Eisenberg, D. (1983) Acta Crystallogr. A39, rms deviations of 2.1 A 813-817. both LDH and MDH (74 out of 80; 16. Jones, T. A. (1978) J. Appl. Cryst. 11, 268-272. and 1.8 A, respectively; coordinates from the Protein Data 17. Konnert, J. H. & Hendrickson, W. A. (1980) Acta Crystallogr. A36, Bank, 1988). The Q-axis dimer formation in LDH and MDH 344-350. is dominated by the association of helices aB, aC, and a3G 18. Richardson, J. S. (1981) Adv. Protein Chem. 34, 167-339. with their twofold related counterparts (36). In GAPDH, even 19. Scrutton, N. S., Berry, A. & Perham, R. N. (1990) Nature (London) though the relative orientations of helices aB and aC are 343, 38-43. slightly different, roughly similar packing is observed at this 20. Rossmann, M. G., Adams, M. J., Buehner, M., Ford, G. C., Hack- ert, M. L., Liljas, A., Rao, S. T., Banaszak, L. J., Hill, E., Tser- interface. Such an association is absent in the 3a,203-HSD noglou, D. & Webb, L. (1973) J. Mol. Biol. 76, 533-537. tetramer where aggregation is profoundly different. The most 21. Ekland, H., Samama, J. P. & Jones, T. A. (1984) Biochemistry 23, significant aspect of the difference is that the a-helices B and 5982-5996. C are on the outer surface of the tetramer. The native dimer 22. Piontek, K., Chakrabarti, P., Schar, H.-P., Rossmann, M. G. & of ADH is stabilized by antiparallel association ofPF strands Zuber, H. (1990) Proteins: Struct. Funct. Genet. 7, 74-92. a twofold similar to the association 23. Matthews, D. A., Alden, R. A., Freer, S. T., Xuong, N. H. & and aA helices about axis, Krout, J. (1979) J. Biol. Chem. 254, 4144-4151. of the P-axis-related 13G strands and aG helices in 3a,20f3- 24. Skarzynski, T., Moody, P. C. E. & Wonacott, A. J. (1987) J. Mol. HSD. Biol. 193, 171-187. Concluding Remarks. The structure determination of 25. Betz, G. & Warren, J. C. (1968) Arch. Biochem. Biophys. 128, 3a,20P-HSD reveals a high degree of similarity to the long- 745-752. chain dehydrogenases in the cofactor binding fold despite no 26. Reddy, B. S., Saenger, W., Muhlegger, K. & Weimann, G. (1981) that J. Am. Chem. Soc. 103, 907-914. significant sequence homology, suggesting they may 27. Declercq, J. P., Germain, G. & Van Meerssche, M. (1972) Cryst. have evolved independently. The subunits of 3a,20l3-HSD Struct. Commun. 1, 13-15. associate in a significantly different way from that ofthe other 28. Pasta, P., Mazzola, G. & Carrea, G. (1987) Biochemistry 26, dehydrogenases. Consistent with the requirement of a tetra- 1247-1251. mer for activity, each of the four catalytic sites is comprised 29. Carrea, G., Pasta, P. & Vecchio, G. (1984) Biochim. Biophys. Acta of atoms from all four subunits. On the basis of the observed 784, 16-23. 30. Grau, U. M., Trommer, W. E. & Rossmann, M. G. (1981) J. Mol. structure, it is possible that the Arg-16 side chain participates Biol. 151, 289-307. in the B-face hydride transfer process, directly or indirectly. 31. Dayhoff, M. O., Barker, W. C. & Hunt, L. T. (1983) Methods The structure determination of 3a,20f3-HSD unequivocally Enzymol. 91, 524-545. demonstrates the presence of a single cofactor site and a 32. Chandrasekhar, K., McPherson, A., Adams, M. J. & Rossmann, single substrate site per subunit and hence confirms the two M. G. (1973) J. Mol. Biol. 76, 503-518. 33. Webb, L. E., Hill, E. J. & Banaszak, L. J. (1973) Biochemistry 12, steroid orientations-one cofactor site model. 5101-5109. 34. Murthy, M. R. N., Garavito, R. M., Johnson, J. E. & Rossmann, We thank Dr. N.-h. Xuong and his group at the Research Resource M. G. (1980) J. Mol. Biol. 138, 859-872. in Protein Crystallography, University of California in San Diego; 35. Eklund, H., Nordstrom, B., Zeppezauer, E., Soderlund, G., Ohls- staff members of the MacCHESS facility, Cornell High Energy son, I., Boiwe, T., Soderberg, B. O., Tapia, O., Branden, C.-I. & Synchrotron Source, and Dr. W. Pangborn (film data collection); Dr. Akeson, A. (1976) J. Mol. Biol. 102, 27-59. J. E. Johnson and his staff at Purdue University (film data process- 36. Rossmann, M. G., Liljas, A., Branden, C.-I. & Banaszak, L. J. ing); the Pittsburgh Supercomputing Center (grant no. DMB890095P (1975) in The Enzymes, ed. Boyer, P. D. (Academic, New York), funded by National Institutes of Health Division of Research Re- Vol. 11A, pp. 61-102. Downloaded by guest on September 28, 2021