Comparison of crystal structures of human type 3 3alpha-hydroxysteroid dehydrogenase reveals an "induced-fit" mechanism and a conserved basic motif involved in the binding of androgen

The aldo-keto reductase (AKR) human type 3 3alpha-hydroxysteroid dehydrogenase (h3alpha-HSD3, AKR1C2) plays a crucial role in the regulation of the intracellular concentrations of testosterone and 5alpha-dihydrotestosterone (5alpha-DHT), two steroids directly linked to the etiology and the progression of many prostate diseases and cancer. This enzyme also binds many structurally different molecules such as 4-hydroxynonenal, polycyclic aromatic hydrocarbons, and indanone. To understand the mechanism underlying the plasticity of its substrate-binding site, we solved the binary complex structure of h3alpha-HSD3-NADP(H) at 1.9 A resolution. During the refinement process, we found acetate and citrate molecules deeply engulfed in the steroid-binding cavity. Superimposition of this structure with the h3alpha-HSD3-NADP(H)-testosterone/acetate ternary complex structure reveals that one of the mobile loops forming the binding cavity operates a slight contraction movement against the citrate molecule while the side chains of many residues undergo numerous conformational changes, probably to create an optimal binding site for the citrate. These structural changes, which altogether cause a reduction of the substrate-binding cavity volume (from 776 A(3) in the presence of testosterone/acetate to 704 A(3) in the acetate/citrate complex), are reminiscent of the "induced-fit" mechanism previously proposed for the aldose reductase, another member of the AKR superfamily. We also found that the replacement of residues Arg(301) and Arg(304), localized near the steroid-binding cavity, significantly affects the 3alpha-HSD activity of this enzyme toward 5alpha-DHT and completely abolishes its 17beta-HSD activity on 4-dione. All these results have thus been used to reevaluate the binding mode of this enzyme for androgens.     

Crystal structure of human type III 3alpha-hydroxysteroid dehydrogenase/bile acid binding protein complexed with NADP(+) and ursodeoxycholate

The crystal structure of human type III 3alpha-hydroxysteroid dehydrogenase (HSD)/bile acid binding protein (AKR1C2) complexed with NADP(+) and 3alpha,7beta-dihydroxy-5beta-cholanic acid (ursodeoxycholate) at 3.0 A resolution is presented. Thus, the three-dimensional structure has now been solved for a human HSD member of the aldo-keto reductase superfamily. AKR1C2 is implicated in the prostatic production of the potent androgen 5alpha-dihydrotestosterone and the hepatic transport of bile acids. It also catalyzes the formation of the neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one in the central nervous system, and its allosteric modulation by fluoxetine has been linked to the use of this drug for premenstrual dsyphoria. Like other members of the superfamily, AKR1C2 folds into an alpha/beta-barrel and binds NADP(+) in an extended conformation. The carboxylate of ursodeoxycholate binds to AKR1C2 in the oxyanion hole at the active site. More interestingly, the orientation of ursodeoxycholate is essentially "backwards" and "upside-down" from that observed for testosterone in the related rat 3alpha-HSD.NADP(+).testosterone ternary complex, where testosterone assumes the position of a 3-ketosteroid substrate. The orientation of ursodeoxycholate is thus similar to that expected of a 17beta-HSD substrate. The ternary structure explains the ability of AKR1C2 to catalyze 3alpha-, 17beta-, and 20alpha-HSD reactions. Comparison of the steroid binding pocket of AKR1C2 with that of rat 3alpha-HSD reveals significant differences in the positions of conserved and nonconserved loop residues, providing insights into the structural basis for the functional flexibility that is observed in all the human 3alpha-HSD isoforms but not in the rat isoform.     

Human 20alpha-hydroxysteroid dehydrogenase: crystallographic and site-directed mutagenesis studies lead to the identification of an alternative binding site for C21-steroids

Human 20alpha-hydroxysteroid dehydrogenase (h20alpha-HSD; AKR1C1) catalyzes the transformation of progesterone (Prog) into 20alpha-hydroxy-progesterone (20alpha-OHProg). Although h20alpha-HSD shares 98% sequence identity with human type 3 3alpha-HSD (h3alpha-HSD3, AKR1C2), these two enzymes differ greatly in their activities. In order to explain these differences, we have solved the crystal structure of h20alpha-HSD in a ternary complex with NADP(+) and 20alpha-OHProg at 1.59A resolution. The steroid is stabilized by numerous hydrophobic interactions and a hydrogen bond between its O20 and the N(epsilon ) atom of His222. This new interaction prevents the formation of a hydrogen bond with the cofactor, as seen in h3alpha-HSD3 ternary complexes. By combining structural, direct mutagenesis and kinetic studies, we found that the H(222)I substitution decreases the K(m) value for the cofactor 95-fold. With these results, we hypothesize that the rotation of the lateral chain of His222 could be a mediating step between the transformation of Prog and the release of the cofactor. Moreover, crystal structure analysis and direct mutagenesis experiments lead us to identify a new residue involved in the binding of Prog. Indeed, the R(304)L substitution leads to a 65-fold decrease in the K(m) value for Prog reduction. We thus propose that Prog is maintained in a new steroid-binding site composed mainly of residues found in the carboxy-terminal region of the protein.     

Structure-function relationships in 3alpha-hydroxysteroid dehydrogenases: a comparison of the rat and human isoforms

3alpha-Hydroxysteroid dehydrogenases (3alpha-HSDs) inactivate steroid hormones in the liver, regulate 5alpha-dihydrotestosterone (5alpha-DHT) levels in the prostate, and form the neurosteroid, allopregnanolone in the CNS. Four human 3alpha-HSD isoforms exist and correspond to AKR1C1-AKR1C4 of the aldo-keto reductase (AKR) superfamily. Unlike the related rat 3alpha-HSD (AKR1C9) which is positional and stereospecific, the human enzymes display varying ratios of 3-, 17-, and 20-ketosteroid reductase activity as well as 3alpha-, 17beta-, and 20alpha-hydroxysteroid oxidase activity. Their k(cat) values are 50-100-fold lower than that observed for AKR1C9. Based on their product profiles and discrete tissue localization, the human enzymes may regulate the levels of active androgens, estrogens, and progestins in target tissues. The X-ray crystal structures of AKR1C9 and AKR1C2 (human type 3 3alpha-HSD, bile acid binding protein and peripheral 3alpha-HSD) reveal that the AKR1C2 structure can bind steroids backwards (D-ring in the A-ring position) and upside down (beta-face inverted) relative to the position of a 3-ketosteroid in AKR1C9 and this may account for its functional plasticity. Stopped-flow studies on both enzymes indicate that the conformational changes associated with binding cofactor (the first ligand) are slow; they are similar in both enzymes but are not rate-determining. Instead the low k(cat) seen in AKR1C2 (50-fold less than AKR1C9) may be due to substrate "wobble" at the plastic active site.     

Mouse 17alpha-hydroxysteroid dehydrogenase (AKR1C21) binds steroids differently from other aldo-keto reductases: identification and characterization of amino acid residues critical for substrate binding

The mouse 17alpha-hydroxysteroid dehydrogenase (m17alpha-HSD) is the unique known member of the aldo-keto reductase (AKR) superfamily able to catalyze efficiently and in a stereospecific manner the conversion of androstenedione (Delta4) into epi-testosterone (epi-T), the 17alpha-epimer of testosterone. Structural and mutagenic studies had already identified one of the residues delineating the steroid-binding cavity, A24, as the major molecular determinant for the stereospecificity of m17alpha-HSD. We report here a ternary complex crystal structure (m17alpha-HSD:NADP(+):epi-T) determined at 1.85 A resolution that confirms this and reveals a unique steroid-binding mode for an AKR enzyme. Indeed, in addition to the interactions found in all other AKRs (van der Waals contacts stabilizing the core of the steroid and the hydrogen bonds established at the catalytic site by the Y55 and H117 residues with the oxygen atom of the ketone group to be reduced), m17alpha-HSD establishes with the other extremity of the steroid nucleus an additional interaction involving K31. By combining direct mutagenesis and kinetic studies, we found that the elimination of this hydrogen bond did not affect the affinity of the enzyme for its steroid substrate but led to a slight but significant increase of its catalytic efficiency (k(cat)/K(m)), suggesting a role for K31 in the release of the steroidal product at the end of the reaction. This previously unobserved steroid-binding mode for an AKR is similar to that adopted by other steroid-binding proteins, the hydroxysteroid dehydrogenases of the short-chain dehydrogenases/reductases (SDR) family and the steroid hormone nuclear receptors. Mutagenesis and structural studies made on the human type 3 3alpha-HSD, a closely related enzyme that shares 73% amino acids identity with the m17alpha-HSD, also revealed that the residue at position 24 of these two enzymes directly affects the binding and/or the release of NADPH, in addition to its role in their 17alpha/17beta stereospecificity.     

Molecular dissection,tissue localization and Ca2+ binding of the ryanodine receptor of Caenorhabditis elegans

We investigated the possible role of residues at the Ccap position in an alpha-helix on protein stability. A set of 431 protein alpha-helices containing a C'-Gly from the Protein Data Bank (PDB) was analyzed, and the normalized frequencies for finding particular residues at the Ccap position, the average fraction of buried surface area, and the hydrogen bonding patterns of the Ccap residue side-chain were calculated. We found that on average the Ccap position is 70% buried and noted a significant correlation (R=0.8) between the relative burial of this residue and its hydrophobicity as defined by the Gibbs energy of transfer from octanol or cyclohexane to water. Ccap residues with polar side-chains are commonly involved in hydrogen bonding. The hydrogen bonding pattern is such that, the longer side-chains of Glu, Gln, Arg, Lys, His form hydrogen bonds with residues distal (>+/-4) in sequence, while the shorter side-chains of Asp, Asn, Ser, Thr exhibit hydrogen bonds with residues close in sequence (<+/-4), mainly involving backbone atoms. Experimentally we determined the thermodynamic propensities of residues at the Ccap position using the protein ubiquitin as a model system. We observed a large variation in the stability of the ubiquitin variants depending on the nature of the Ccap residue. Furthermore, the measured changes in stability of the ubiquitin variants correlate with the hydrophobicity of the Ccap residue. The experimental results, together with the statistical analysis of protein structures from the PDB, indicate that the key hydrophobic capping interactions between a helical residue (C3 or C4) and a residue outside the helix (C", C3' or C4') are frequently enhanced by the hydrophobic interactions with Ccap residues.     

Inhibition of 20 alpha-hydroxysteroid dehydrogenase activity by follicle-stimulating hormone and androgens in cultured rat granulosa cells: a search for the mechanism of action

Alterations of progesterone metabolism and especially of 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity were studied in cultured rat granulosa cells following various treatments. The cells were incubated for up to 48 h with or without follicle-stimulating hormone (FSH), androgens, hydroxyflutamide, estrogens, chlorea toxin, and dibutyryl cAMP [Bu2 cAMP]. Subsequently, the cells were incubated for 3 h with [4-14 C] progesterone (0.5 microM). The progesterone utilization and accumulation of 20 alpha-reduced and 5 alpha-reduced metabolites were assessed following thin-layer chromatography separation of radiolabeled steroids. Both FSH (1 microgram/ml) and testosterone (0.5 microM) decreased the 20 alpha-HSD activity by decreasing the maximal velocity (by 52% and 37%, respectively) without changing significantly the Km value. The inhibition of 20 alpha-HSD was demonstrable following 12 and 24 h exposure to FSH and following 24 and 48 h exposure to testosterone. Effects comparable to that induced by testosterone were elicited by other androgens (androstenedione and 5 alpha-dihydrotestosterone), but not by estrogens (estradiol-17 beta and estrone). Hydroxyflutamide reversed testosterone-induced effects: the increase of endogenous progesterone accumulation and the decrease of 20 alpha-HSD activity. Both cholera toxin (0.001-10 micrograms/ml) and Bu2 cAMP (62.5-1000 micrograms/ml) caused a dose-dependent inhibition of 20 alpha-HSD activity. Present results indicate that: the inhibition of 20 alpha-HSD by both FSH and androgens may be of a noncompetitive nature; androgen action on 20 alpha-HSD may be a true androgenic, receptor-mediated effect; and cAMP may mediate the FSH action on 20 alpha-HSD activity.     

Crystal structures of mouse 17alpha-hydroxysteroid dehydrogenase (apoenzyme and enzyme-NADP(H) binary complex): identification of molecular determinants responsible for the unique 17alpha-reductive activity of this enzyme

Very recently, the mouse 17alpha-hydroxysteroid dehydrogenase (m17alpha-HSD), a member of the aldo-keto reductase (AKR) superfamily, has been characterized and identified as the unique enzyme able to catalyze efficiently and in a stereospecific manner the conversion of androstenedione (Delta4) into epitestosterone (epi-T), the 17alpha-epimer of testosterone. Indeed, the other AKR enzymes that significantly reduce keto groups situated at position C17 of the steroid nucleus, the human type 3 3alpha-HSD (h3alpha-HSD3), the human and mouse type 5 17beta-HSD, and the rabbit 20alpha-HSD, produce only 17beta-hydroxy derivatives, although they possess more than 70% amino acid identity with m17alpha-HSD. Structural comparisons of these highly homologous enzymes thus offer an excellent opportunity of identifying the molecular determinants responsible for their 17alpha/17beta-stereospecificity. Here, we report the crystal structure of the m17alpha-HSD enzyme in its apo-form (1.9 A resolution) as well as those of two different forms of this enzyme in binary complex with NADP(H) (2.9 A and 1.35 A resolution). Interestingly, one of these binary complex structures could represent a conformational intermediate between the apoenzyme and the active binary complex. These structures provide a complete picture of the NADP(H)-enzyme interactions involving the flexible loop B, which can adopt two different conformations upon cofactor binding. Structural comparison with binary complexes of other AKR1C enzymes has also revealed particularities of the interaction between m17alpha-HSD and NADP(H), which explain why it has been possible to crystallize this enzyme in its apo form. Close inspection of the m17alpha-HSD steroid-binding cavity formed upon cofactor binding leads us to hypothesize that the residue at position 24 is of paramount importance for the stereospecificity of the reduction reaction. Mutagenic studies have showed that the m17alpha-HSD(A24Y) mutant exhibited a completely reversed stereospecificity, producing testosterone only from Delta4, whereas the h3alpha-HSD3(Y24A) mutant acquires the capacity to metabolize Delta4 into epi-T.     

Mutational studies using a cation-conducting GABAA receptor reveal the selectivity determinants of the Cys-loop family of ligand-gated ion channels

The present study evaluates how four key amino acid residue positions (- 4' to - 1') within the M1-M2 linker of the GABA(A) receptor beta subunit influences ion selectivity of a cation-conducting GABA receptor. Cation selectivity was found to be highly dependent on the side-chains of the amino acid residues present. The critical factor for cation selectivity was the presence of a negatively charged Glu or Asp residue in the -1' position. Receptors containing the neutral amino acids Gln or Asn or a positively charged Arg residue were anion selective. In the presence of a -1' Glu residue, the amino acids in adjacent positions were also found to be important determinants of cation selectivity. Moreover, the length of the M1-M2 linker as well as the presence of a Pro residue within this segment also affected ion selectivity, suggesting that the local environment and three-dimensional position of the -1' Glu are essential determinants of cation permeation. Conversely, no specific amino acid residues were found to be essential for anion selectivity, suggesting that the basic architecture of the selectivity segment of this class of receptor channels is optimally suited for anion conduction.     

Functional expression, purification, and characterization of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni

3alpha-Hydroxysteroid dehydrogenase (3alpha-HSD) catalyzes the oxidoreduction at carbon 3 of steroid hormones and is postulated to initiate the complete mineralization of the steroid nucleus to CO(2) and H(2)O in Comamonas testosteroni. By this activity, 3alpha-HSD provides the basis for C. testosteroni to grow on steroids as sole carbon and energy source. 3alpha-HSD was cloned and overexpressed in E. coli and purified to homogeneity by an affinity chromatography system as His-tagged protein. The recombinant enzyme was found to be functional as oxidoreductase toward a variety of steroid substrates, including androstanedione, 5alpha-dihydrotestosterone, androsterone, cholic acid, and the steroid antibiotic fusidic acid. The enzyme also catalyzes the carbonyl reduction of nonsteroidal aldehydes and ketones such as metyrapone, p-nitrobenzaldehyde and a novel insecticide (NKI 42255), and, based on this pluripotent substrate specificity, was named 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR). It is suggested that 3alpha-HSD/CR contributes to important defense strategies of C. testosteroni against natural and synthetic toxicants. Antibodies were generated in rabbits against the entire 3alpha-HSD/CR protein, and may now be used for evaluating the pattern of steroid induction in C. testosteroni on the protein level. Upon gel permeation chromatography the purified enzyme elutes as a 49.4 kDa protein revealing for the first time the dimeric nature of 3alpha-HSD/CR of C. testosteroni.     

Molecular characterization of the cynomolgus monkey Macaca fascicularis steroidogenic enzymes belonging to the aldo-keto reductase family

Steroidogenic enzymes belonging to the aldo-keto reductase family (AKR) possess highly homologous sequences while having different activities. To gain further knowledge about the function as well as the regulation of these enzymes in the monkey, we have isolated cDNA sequences encoding monkey type 5 17beta-hydroxysteroid dehydrogenase, 20alpha-hydroxysteroid dehydrogenase and 3alpha-hydroxysteroid dehydrogenase, and characterized their enzymatic activity and mRNA tissue distribution. Sequence analysis indicates that these enzymes share approximately 94 and 76% amino acid identity with human and mouse homologs, respectively. Monkey type 5 17beta-HSD possesses 95.9% amino acid sequence identity with human type 5 17beta-HSD. It catalyzes the transformation of 4-androstenedione into testosterone, but it lacks 20alpha-hydroxysteroid dehydrogenase activity that is present in the human enzyme. This activity seems to be specific to human, since mouse type 5 17beta-HSD does not show significant 20alpha-HSD activity. In addition, monkey and mouse 20alpha-HSD possess relatively high 20alpha-, 3alpha-, and 17beta-HSD activities, while their human counterpart is confined to 20alpha-HSD activity. The monkey 3alpha-HSD possesses relatively high 3alpha-, 17beta-, and 20alpha-HSD activities; human type 1 3alpha-HSD exerts 3alpha- and 20alpha-HSD activities; the mouse 3alpha-HSD displays a unique 3alpha-HSD activity. Quantification of mRNA expression shows that the monkey 3alpha-HSD is exclusively expressed in the liver, while the type 5 17beta-HSD is predominately found in the kidney, with lower levels observed in the stomach, liver, and colon. Monkey 20alpha-HSD mRNA is highly expressed in the kidney, stomach, and liver. Our study provides the basis for future investigations on the regulation and function of these enzymes in the monkey.     

Engineering steroid hormone specificity into aldo-keto reductases

Steroid hormone transforming aldo-keto reductases (AKRs) include virtually all mammalian 3alpha-hydroxysteroid dehydrogenases (3alpha-HSDs), 20alpha-HSDs, as well as the 5beta-reductases. To elucidate the molecular determinants of steroid hormone recognition we used rat liver 3alpha-HSD (AKR1C9) as a starting structure to engineer either 5beta-reductase or 20alpha-HSD activity. 5beta-Reductase activity was introduced by a single point mutation in which the conserved catalytic His (H117) was mutated to Glu117. The H117E mutant had a k(cat) comparable to that for homogeneous rat and human liver 5beta-reductases. pH versus k(cat) profiles show that this mutation increases the acidity of the catalytic general acid Tyr55. It is proposed that the increased TyrOH(2)(+) character facilitates enolization of the Delta(4)-3-ketosteroid and subsequent hydride transfer to C5. Since 5beta-reductase precedes 3alpha-HSD in steroid hormone metabolism it is likely that this metabolic pathway arose by gene duplication and point mutation. 3alpha-HSD is positional and stereospecific for 3-ketosteroids and inactivates androgens. The enzyme was converted to a robust 20alpha-HSD, which is positional and stereospecific for 20-ketosteroids and inactivates progesterone, by the generation of loop-chimeras. The shift in log(10)(k(cat)/K(m)) from androgens to progestins was of the order of 10(11). This represents a rare example of how steroid hormone specificity can be changed at the enzyme level. Protein engineering with predicted outcomes demonstrates that the molecular determinants of steroid hormone recognition in AKRs will be ultimately rationalized.     

Molecular determinants of steroid recognition and catalysis in aldo-keto reductases. Lessons from 3alpha-hydroxysteroid dehydrogenase.

Hydroxysteroid Dehydrogenases (HSDs) regulate the occupancy of steroid hormone receptors by converting active steroid hormones into their cognate inactive metabolites. HSDs belong to either the Short-chain Dehydrogenase/Reductases (SDRs) or the Aldo-Keto Reductases (AKRs). The AKRs include virtually all mammalian 3alpha-HSDs, Type 5 17beta-HSD, ovarian 20alpha-HSDs as well as the steroid 5beta-reductases. Selective inhibitors of 3alpha-HSD isoforms could control occupancy of the androgen and GABA(A) receptors, while broader based AKR inhibitors targeting 3alpha-HSD, 20alpha-HSD and prostaglandin F2alpha synthase could maintain pregnancy. We have determined three X-ray crystal structures of rat liver 3alpha-HSD, a representative AKR. These structures are of the apoenzyme (E), the binary-complex (E.NADP-), and the ternary complex (E.NADP+.testosterone). These structures are being used with site-directed mutagenesis to define the molecular determinants of steroid recognition and catalysis as a first step in rational inhibitor design. A conserved catalytic tetrad (Tyr55, Lys84, His117 and Asp50) participates in a 'proton-relay' in which Tyr55 acts as general acid/base catalyst. Its bifunctionality relies on contributions from His117 and Lys84 which alter the pKb and pKa, respectively of this residue. Point mutation of the tetrad results in different enzymatic activities. H117E mutants display 5beta-reductase activity while Y55F and Y55S mutants retain quinone reductase activity. Our results suggest that different transition states are involved in these reaction mechanisms. The ternary complex structure shows that the mature steroid binding pocket is comprised of ten residues recruited from five loops, and that there is significant movement of a C-terminal loop on binding ligand. Mutagenesis of pocket tryptophans shows that steroid substrates and classes of nonsteroidal inhibitors exhibit different binding modes which may reflect ligand-induced loop movement. Exploitation of these findings using steroidal and nonsteroidal mechanism based inactivators may lead to selective and broad based AKR inhibitors.     

A model on the regulation of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase expression in Comamonas testosteroni

3alpha-Hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) from Comamonas testosteroni is a key enzyme involved in the degradation of steroids and xenobiotic carbonyl compounds. The enzyme has recently been cloned and characterized by our group. A strong induction of enzyme activity is observed in the presence of steroids like testosterone. In the present investigation, two repressor proteins (Rep1 and Rep2) containing 78 and 420 amino acids, respectively, were found to regulate 3alpha-HSD/CR gene (hsdA) expression. Gel shift experiments showed that Rep2 binds to a 10 nucleotide sequence 9 bp upstream of the hsdA promoter. The deletion of this cis-regulating sequence significantly increases hsdA expression. About 1633 bp further upstream, a second ten nucleotide sequence, complementary to the first one, was found, which is also recognized by Rep2 and increases hsdA expression, if deleted. To purify the repressor proteins, the genes encoding each were cloned into His-tag expression vectors and overexpressed in Escherichia coli. Rep1 does not bind to DNA but may bind to 3alpha-HSD/CR mRNA as predicted by its secondary structure. Concluding from our data, induction of 3alpha-HSD/CR in C. testosteroni by steroids in fact appears to be a de-repression, where the steroidal 'inducer' prevents the binding of the two repressor proteins to the hsdA promoter and mRNA, respectively.     

Comprehensive functional maps of the antigen-binding site of an anti-ErbB2 antibody obtained with shotgun scanning mutagenesis

Shotgun scanning combinatorial mutagenesis was used to study the antigen-binding site of Fab2C4, a humanized monoclonal antibody fragment that binds to the extracellular domain of the human oncogene product ErbB2. Essentially all the residues in the Fab2C4 complementarity determining regions (CDRs) were alanine-scanned using phage-displayed libraries that preferentially allowed side-chains to vary as the wild-type or alanine. A separate homolog-scan was performed using libraries that allowed side-chains to vary only as the wild-type or a similar amino acid residue. Following binding selections to isolate functional clones, DNA sequencing was used to determine the wild-type/mutant ratios at each varied position, and these ratios were used to assess the contributions of each side-chain to antigen binding. The alanine-scan revealed that most of the side-chains that contribute to antigen binding are located in the heavy chain, and the Fab2C4 three-dimensional structure revealed that these residues fall into two groups. The first group consists of solvent-exposed residues which likely make energetically favorable contacts with the antigen and thus comprise the functional-binding epitope. The second group consists of buried residues with side-chains that pack against other CDR residues and apparently act as scaffolding to maintain the functional epitope in a binding-competent conformation. The homolog-scan involved subtle mutations, and as a result, only a subset of the side-chains that were intolerant to alanine substitutions were also intolerant to homologous substitutions. In particular, the 610 A2 functional epitope surface revealed by alanine-scanning shrunk to only 369 A2 when mapped with homologous substitutions, suggesting that this smaller subset of side-chains may be involved in more precise contacts with the antigen. The results validate shotgun scanning as a rapid and accurate method for determining the functional contributions of individual side-chains involved in protein-protein interactions.     

Microsomal 20 alpha-hydroxysteroid dehydrogenase activity for progesterone in human placenta

Placental 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity was studied in order to evaluate the mechanism of continuation of pregnancy and initiation of labor. The placentas obtained at various gestational weeks were homogenized and fractionated into "nuclear", "mitochondrial", "microsomal" and "supernatant" fractions. Each fraction was incubated with 14C-progesterone and a hydrogen donor. Enzymatic activity was measured by the conversion of progesterone to 20 alpha-dihydroprogesterone. The highest activity of 20 alpha-HSD for progesterone was found to be localized in "microsomal" fraction. The Km constant of 20 alpha-HSD was 4.5 X 10(-6)M for progesterone in "microsomal" fraction. It was found that placental microsomal 20 alpha-HSD required NADPH as well as NADH. 20 alpha-HSD activity for progesterone increased as gestational weeks advanced. The addition of DHA-sulfate and DHA inhibited 20 alpha-HSD activity for progesterone significantly, suggesting that the steroid produced by the feto-placental unit may be involved in the metabolism of progesterone in human placenta.