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).