The first crystal structure of a gramicidin complex with sodium: high-resolution study of a nonstoichiometric gramicidin D-NaI complex.

The crystal structure of the nonstoichiometric complex of gramicidin D with NaI has been studied using synchrotron radiation at 100 K. The limiting resolution was 1.25 A and the R factor was 16% for 19 883 observed reflections. The general architecture of the antiparallel two-stranded gramicidin dimers in the studied crystal was a right-handed antiparallel double-stranded form that closely resembles the structures of other right-handed species published to date. However, there were several surprising observations. In addition to the significantly different composition of linear gramicidins identified in the crystal structure, including the absence of the gramicidin C form, only two cationic sites were found in each of the two independent dimers (channels), which were partially occupied by sodium, compared with the seven sites found in the RbCl complex of gramicidin. The sum of the partial occupancies of Na(+) was only 1.26 per two dimers and was confirmed by the similar content of iodine ions (1.21 ions distributed over seven sites), which was easily visible from their anomalous signal. Another surprising observation was the significant asymmetry of the distributions and occupancies of cations in the gramicidin dimers, which was in contrast to those observed in the high-resolution structures of the complexes of heavier alkali metals with gramicidin D, especially that of rubidium.

Nonstoichiometric complex of gramicidin D with KI at 0.80 A resolution.

The crystal structure of a nonstoichiometric complex of gramicidin D (gD) with KI has been determined at 100 K using synchrotron radiation. The final R factor was 0.106 for 83 988 observed reflections (Friedel pairs were not merged) collected to 0.80 A. The structure consists of four independent pentadecapeptides and numerous solvent molecules and salt ions. The general architecture of the antiparallel double-stranded gramicidin dimers in the crystal (a right-handed antiparallel DSbetaH(R) form) closely resembles that of previously published cation complexes of gD. However, a significantly different mixture of gramicidin isomers is found in the crystal of the KI complex, including partial occupancy of phenylalanine at position 11. Only three sites in each of the two crystallographically independent channels are partially occupied by potassium cations instead of the commonly observed seven sites. The sum of the partial occupancies of K(+) (1.10 per two dimers) is consistent with the sum of the iodide occupancies (1.095 over eight sites), which is also confirmed by the anomalous signal of the iodide. There was a significant asymmetry of the distribution and occupancies of cations in the crystallographically independent gramicidin channels, in contrast to the distribution found in the rubidium chloride complex with gD.

Structure of gramicidin D-RbCl complex at atomic resolution from low-temperature synchrotron data: interactions of double-stranded gramicidin channel contents and cations with channel wall.

Gramicidin D (gD) is a naturally occurring ionophoric antibiotic that forms membrane channels specific for monovalent cations. The crystal structure of the RbCl complex of gD has been determined at 1.14 A resolution from low-temperature (100 K) synchrotron-radiation data with a final R of 16%. The structure was refined with anisotropic temperature factors for all non-H atoms and with partial occupancies for many of them. The asymmetric unit in the crystal contains four crystallographically independent molecules that form two right-handed antiparallel double-stranded dimers. There are seven distinct rubidium-binding sites in each dimeric channel. The occupancy factors of Rb cations are between 0.11 and 0.35 and the total ion contents of the two crystallographically independent channels are 1.59 and 1.22 ions, respectively. Although each channel is 'chemically symmetrical', the side-chain conformations, the distributions of rubidium cations and their binding sites in the two independent channels are not. Cations are 'coordinated' by delocalized pi-electrons of three to five carbonyl groups that together with peptide backbone chains form the gramicidin channel walls. The water:cation ratio in the channel interior is four or five:one, and five or six waters separate Rb cations during their passage through the channel.

Serine 124 completes the Tyr, Lys and Ser triad responsible for the catalysis of human type 1 3beta-hydroxysteroid dehydrogenase.

Human 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) is a key steroidogenic enzyme that catalyzes the first step in the conversion of circulating dehydroepiandrosterone (DHEA), pregnenolone or 17alpha-hydroxypregenolone to produce the appropriate, active steroid hormone(s): estradiol, testosterone, progesterone, aldosterone or cortisol respectively. Our mutagenesis studies have identified Tyr154 and Lys158 as catalytic residues for the 3beta-HSD reaction. Our three-dimensional homology model of 3beta-HSD shows that Tyr154 and Lys158 are oriented near the 3beta-hydroxyl group of the bound substrate steroid, and predicts that Ser123 or Ser124 completes a Tyr-Lys-Ser catalytic triad that operates in many other dehydrogenases. The S123A and S124A mutants of human type 1 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD1) were created by PCR-based mutagenesis, expressed in insect cells using baculovirus and purified to homogeneity. The S124A mutant exhibits no 3beta-HSD activity and has a K(m) value (83.6 microM) for the isomerase substrate that is threefold greater than that of wild-type 1 isomerase. In contrast, S123A has substantial 3beta-HSD activity (DHEA K(m)=11.2 microM; k(cat)=0.8 min(-1)) and utilizes isomerase substrate, 5-androstene-3,17-dione, with a K(m) value (27.6 microM) that is almost identical to wild-type. The K(m) value (4.3 microM) of S124A for NADH as an allosteric activator of isomerase is similar to that of the wild-type 1 enzyme, indicating that Ser124 is not involved in cofactor binding. S123A utilizes NAD as a cofactor for 3beta-HSD and NADH as the activator for isomerase with K(m) values that are similar to wild-type. The 3beta-HSD activities of S123A and wild-type 3beta-HSD increase by 2.7-fold when the pH is raised from 7.4 to the optimal pH 9.7, but S124A exhibits very low residual 3beta-HSD activity that is pH-independent. These kinetic analyses strongly suggest that the Ser124 residue completes the catalytic triad for the 3beta-HSD activity. Since there are 29 Ser residues in the primary structure of human 3beta-HSD1, our homology model of the catalytic domain has been validated by this accurate prediction. A role for Ser124 in the binding of the isomerase substrate, which is the 3beta-HSD product-steroid of the bifunctional enzyme protein, is also suggested. These observations further characterize the structure/function relationships of human 3beta-HSD and bring us closer to the goal of selectively inhibiting the type 1 enzyme in placenta to control the timing of labor or in hormone-sensitive breast tumors to slow their growth.

Crystal structure of an anti-interleukin-2 monoclonal antibody Fab complexed with an antigenic nonapeptide.

The three-dimensional structure of the Fab fragment of a monoclonal antibody (LNKB-2) to human interleukin-2 (IL-2) complexed with a synthetic antigenic nonapeptide, Ac-Lys-Pro-Leu-Glu-Glu-Val-Leu-Asn-Leu-OMe, has been determined at 3.0 A resolution. In the structure, four out of the six hypervariable loops of the Fab (complementarity determining regions [CDRs] L1, H1, H2, and H3) are involved in peptide association through hydrogen bonding, salt bridge formation, and hydrophobic interactions. The Tyr residues in the Fab antigen binding site play a major role in antigen-antibody recognition. The structures of the complexed and uncomplexed Fab were compared. In the antigen binding site the CDR-L1 loop of the antibody shows the largest structural changes upon peptide binding. The peptide adopts a mostly alpha-helical conformation similar to that in the epitope fragment 64-72 of the IL-2 antigen. The side chains of residues Leu 66, Val 69, and Leu 70, which are shielded internally in the IL-2 structure, are involved in interactions with the Fab in the complex studied. This indicates that antibody-antigen complexation involves a significant rearrangement of the epitope-containing region of the IL-2 with retention of the alpha-helical character of the epitope fragment.

Porcine carbonyl reductase. structural basis for a functional monomer in short chain dehydrogenases/reductases.

Porcine testicular carbonyl reductase (PTCR) belongs to the short chain dehydrogenases/reductases (SDR) superfamily and catalyzes the NADPH-dependent reduction of ketones on steroids and prostaglandins. The enzyme shares nearly 85% sequence identity with the NADPH-dependent human 15-hydroxyprostaglandin dehydrogenase/carbonyl reductase. The tertiary structure of the enzyme at 2.3 A reveals a fold characteristic of the SDR superfamily that uses a Tyr-Lys-Ser triad as catalytic residues, but exhibits neither the functional homotetramer nor the homodimer that distinguish all SDRs. It is the first known monomeric structure in the SDR superfamily. In PTCR, which is also active as a monomer, a 41-residue insertion immediately before the catalytic Tyr describes an all-helix subdomain that packs against interfacial helices, eliminating the four-helix bundle interface conserved in the superfamily. An additional anti-parallel strand in the PTCR structure also blocks the other strand-mediated interface. These novel structural features provide the basis for the scaffolding of one catalytic site within a single molecule of the enzyme.

[Spatial structure of a Fab-fragment of a monoclonal antibody to human interleukin-2 in two crystalline forms at a resolution of 2.2 and 2.9 angstroms]. / Prostranstvennaia struktura Fab-fragmenta monoklonal'nogo antitela k interleikinu-2 cheloveka v dvukh kristallicheskikh formakh pri razreshenii 2.2 i 2.9 A.

The three-dimensional structure of the antigen-binding fragment of a monoclonal antibody to human interleukin-2 was determined in two crystal forms by the X-ray method of molecular replacement at 2.2 and 2.9 A resolutions. The spatial structure of the protein and the stereochemistry of its antigen-binding site were analyzed.

Crystal structure of bovine duodenase, a serine protease, with dual trypsin and chymotrypsin-like specificities.

The three-dimensional structure of duodenase, a serine protease from bovine duodenum mucosa, has been determined at 2.4A resolution. The enzyme, which has both trypsin-like and chymotrypsin-like activities, most closely resembles human cathepsin G with which it shares 57% sequence identity and similar specificity. The catalytic Ser195 in duodenase adopts the energetically favored conformation typical of serine proteinases and unlike the strained state typical of lipase/esterases. Of several waters in the active site of duodenase, the one associated with Ser214 is found in all serine proteinases and most lipase/esterases. The conservation of the Ser214 residue in serine proteinase, its presence in the active site, and participation in a hydrogen water network involving the catalytic triad (His57, Asp107, and Ser195) argues for its having an important role in the mechanism of action. It may be referred to as a fourth member of the catalytic triad. Duodenase is one of a growing family of enzymes that possesses trypsin-like and chymotrypsin-like activity. Not long ago, these activities were considered to be mutually exclusive. Computer modeling reveals that the S1 subsite of duodenase has structural features compatible with effective accommodation of P1 residues typical of trypsin (Arg/Lys) and chymotrypsin (Tyr/Phe) substrates. The determination of structural features associated with functional variation in the enzyme family may permit design of enzymes with a specific ratio of trypsin and chymotrypsin activities.

Steroid dehydrogenase structures, mechanism of action, and disease.

Steroid dehydrogenase enzymes influence mammalian reproduction, hypertension, neoplasia, and digestion. The three-dimensional structures of steroid dehydrogenase enzymes reveal the position of the catalytic triad, a possible mechanism of keto-hydroxyl interconversion, a molecular mechanism of inhibition, and the basis for selectivity. Glycyrrhizic acid, the active ingredient in licorice, and its metabolite carbenoxolone are potent inhibitors of human 11 beta-hydroxysteroid dehydrogenase and bacterial 3 alpha, 20 beta-hydroxysteroid dehydrogenase (3 alpha, 20 beta-HSD). The three-dimensional structure of the 3 alpha, 20 beta-HSD carbenoxolone complex unequivocally verifies the postulated active site of the enzyme, shows that inhibition is a result of direct competition with the substrate for binding, and provides a plausible model for the mechanism of inhibition of 11 beta-hydroxysteroid dehydrogenase by carbenoxolone. The structure of the ternary complex of human 17 beta-hydroxysteroid dehydrogenase type 1 (17 beta-HSD) with the cofactor NADP+ and the antiestrogen equilin reveals the details of binding of an inhibitor in the active site of the enzyme and the possible roles of various amino acids in the catalytic cleft. The short-chain dehydrogenase reductase (SDR) family includes these steroid dehydrogenase enzymes and more than 60 other proteins from human, mammalian, insect, and bacterial sources. Most members of the family contain the tyrosine and lysine of the catalytic triad in a YxxxK sequence. X-ray crystal structures of 13 members of the family have been completed. When the alpha-carbon backbone of the cofactor binding domains of the structures are superimposed, the conserved residues are at the core of the structure and in the cofactor binding domain, but not in the substrate binding pocket.

[Isolation, crystallization, and preliminary x-ray study of a Fab fragment of monoclonal antibody against human interleukin-2 and its complex with antigenic peptide]. / Poluchenie, kristallizatsiia i predvaritel'nye rentgenostrukturnye issledovaniia Fab-fragmenta monoklonal'nogo antitela k interleikinu-2 cheloveka i ego kompleksa s antigennym peptidom.

Antigen-binding fragments (Fab) of mouse monoclonal antibodies to human interleukin-2 were obtained in preparative quantities by a modified procedure. These Fab-fragments were shown to be homogeneous according to the isoelectric focusing method. Various monocrystals of these free Fab-fragments and their complexes with the antigenic peptide corresponding to the 59-72 sequence of interleukin-2 were obtained. These were shown to be suitable for X-ray and were preliminarily studied by X-ray.

Structure of Ustilago maydis killer toxin KP6 alpha-subunit. A multimeric assembly with a central pore.

Ustilago maydis is a fungal pathogen of maize, some strains of which secrete killer toxins. The toxins are encoded by double-stranded RNA viruses in the cell cytoplasm. The U. maydis killer toxin KP6 contains two polypeptide chains, alpha and beta, having 79 and 81 amino acids, respectively, both of which are necessary for its killer activity. The crystal structure of the alpha-subunit of KP6 (KP6alpha) has been determined at 1.80-A resolution. KP6alpha forms a single domain structure that has an overall shape of an ellipsoid with dimensions 40 A x 26 A x 21 A and belongs to the alpha/beta-sandwich family. The tertiary structure consists of a four-stranded antiparallel beta-sheet, a pair of antiparallel alpha-helices, a short strand along one edge of the sheet, and a short N-terminal helix. Although the fold is reminiscent of toxins of similar size, the topology of KP6alpha is distinctly different in that the alpha/beta-sandwich motif has two right-handed betaalphabeta split crossovers. Monomers of KP6alpha assemble through crystallographic symmetries, forming a hexamer with a central pore lined by hydrophobic N-terminal helices. The central pore could play an important role in the mechanism of the killing action of the toxin.

Gramicidin D conformation, dynamics and membrane ion transport.

The linear pentadecapeptide antibiotic, gramicidin D, a heterogeneous mixture of six components, is a naturally occurring product of Bacillus brevis known to form ion channels in synthetic and natural membranes. The conformation of gramicidin A in the solid state, in organic solvents, and in planar lipid bilayers and the relationship between the composition and the conformation of gramicidin and its selective transport of ions across membranes has been the subject of intense investigation for over 50 years. The x-ray crystal structure and nmr solution spectroscopy agree fully with one another and reveal that entirely different conformations of gramicidin are present in uncomplexed and ion complexed forms. Precise refinements of the three-dimensional structures of naturally occurring gramicidin D in crystals obtained from methanol, ethanol, and n-propanol demonstrate the unexpected presence of stable left-handed antiparallel double-helical heterodimers that vary with the crystallization solvent. The side chains of Trp residues in the three structures exhibit sequence-specific patterns of conformational preference. Tyr substitution for Trp at position 11 appears to favor beta ribbon formation and stabilization of the antiparallel double helix. This conformation acts as a template for gramicidin folding and nucleation of the different crystal forms. The fact that a minor component in a heterogeneous mixture influences aggregation and crystal nucleation has potential applications to other systems in which anomalous behavior is exhibited by aggregation of apparently homogeneous materials, such as the enigmatic behavior of prion proteins. The crystallographically determined structures of cesium, potassium, rubidium, and hydronium ion complexes of gramicidin A are in excellent agreement with the nmr structure determination of the cesium ion gramicidin complex in a methanol chloroform mixture (50 : 50). The right-handed antiparallel double stranded double helical structures (DSDHR) also exhibit geometric features compatible with the solid-state 15N and 2H nmr data recorded for gramicidin in planar lipid bilayers and attributed to the active form of gramicidin A. The DSDHR crystal structures reveal an ion channel with a single partially solvated cation distributed over three ion binding sites. The channel lumen is relatively smooth and electrostatically negative as required for cation passage, while the exterior is electrostatically neutral, a requirement for membrane insertion. The "coordination" of the Cs+ ion is achieved by interaction with the pi orbitals of the carbonyls which do not point toward the ions. The K+ binding sites, which are similar in position to Cs+ binding sites, are shifted off center slightly toward the wall of the channel.

Heterodimer formation and crystal nucleation of gramicidin D.

The linear pentadecapeptide antibiotic gramicidin D is a heterogeneous mixture of six components. Precise refinements of three-dimensional structures of naturally occurring gramicidin D in crystals obtained from methanol, ethanol, and n-propanol demonstrate the unexpected presence of stable left-handed antiparallel double-helical heterodimers that vary with the crystallization solvent. The side chains of Trp residues in the three structures exhibit sequence-specific patterns of conformational preference. Tyr substitution for Trp at position 11 appears to favor beta ribbon formation and stabilization of the antiparallel double helix that acts as a template for gramicidin folding and nucleation of different crystal forms. The fact that a minor component in a heterogeneous mixture influences aggregation and crystal nucleation has potential applications to other systems in which anomalous behavior is exhibited by aggregation of apparently homogeneous materials, such as the enigmatic behavior of prion proteins.

The conducting form of gramicidin A is a right-handed double-stranded double helix.

The linear pentadecapeptide antibiotic, gramicidin D, is a naturally occurring product of Bacillus brevis known to form ion channels in synthetic and natural membranes. The x-ray crystal structures of the right-handed double-stranded double-helical dimers (DSDH) reported here agree with 15N-NMR and CD data on the functional gramicidin D channel in lipid bilayers. These structures demonstrate single-file ion transfer through the channels. The results also indicate that previous crystal structure reports of a left-handed double-stranded double-helical dimer in complex with Cs+ and K+ salts may be in error and that our evidence points to the DSDH as the major conformer responsible for ion transport in membranes.

Acetyl xylan esterase II from Penicillium purpurogenum is similar to an esterase from Trichoderma reesei but lacks a cellulose binding domain.

Penicillium purpurogenum produces at least two acetyl xylan esterases (AXE I and II). The AXE II cDNA, genomic DNA and mature protein sequences were determined and show that the axe 2 gene contains two introns, that the primary translation product has a signal peptide of 27 residues, and that the mature protein has 207 residues. The sequence is similar to the catalytic domain of AXE I from Trichoderma reesei (67% residue identity) and putative active site residues are conserved, but the Penicillium enzyme lacks the linker and cellulose binding domain, thus explaining why it does not bind cellulose in contrast to the Trichoderma enzyme. These results point to a possible common ancestor gene for the active site domain, while the linker and the binding domain may have been added to the Trichoderma esterase by gene fusion.

Structure and mechanism of action and inhibition of steroid dehydrogenase enzymes involved in hypertension.

Members of the NADPH-dependent short chain dehydrogenase/reductase (SDR) family control blood pressure, fertility, and natural and neoplastic growth. Despite the fact that only one amino acid residue is strictly conserved in the 100 known members of the family, all appear to have a dinucleotide-binding Rossmann fold and homologous catalytic residues including the conserved tyrosine. Variation in the binding pocket creates specificity for steroids, prostaglandins, sugars and alcohols. The critically important tyrosine appears to maintain a fixed position relative to the scaffolding of the Rossmann fold and the cofactor position, while the substrate-binding pocket alters in such a way that the dehydrogenation/reduction reaction site is brought into bonding distance of the tyrosine hydroxyl group. Licorice induces high blood pressure by inhibiting an SDR in the kidney. The crystal structure of the complex of 3alpha,20beta-hydroxysteroid dehydrogenase and carbenoxolone reveals the mechanism of enzyme inhibition by licorice. The most potent dehydrogenase enzyme inhibitors are those that displace substrate and cofactor and form strong hydrogen bonds to one or more amino acid residues involved in catalysis.

Subcellular localization, substrate specificity and crystallization of duodenase, a potential activator of enteropeptidase.

Duodenase, a serine protease from bovine duodenum mucosa, was located in endoplasmic reticulum, the Golgi secretory granules of epithelial cells and ducts of Brunner's glands by the A-gold immunocytochemical method. Duodenase exhibits trypsin-like and chymotrypsin-like specificities with a preference for substrates having lysine at the P1 and proline at the P2 positions. The kinetic constants for the hydrolysis of 21 potential duodenase substrates are reported. The best substrates were found to be alpha-N-tosylglycylprolyllysine 4-nitroanilide (k[cat]/Km of 35000 M[-1] s[-1]), alpha-N-succinylthreonylprolyllysine 4-nitroanilide (k[cat]/Km of 18000 M[-1] s[-1]) and alpha-N-serylprolyllysine 4-nitroanilide (k[cat]/Km of 2600 m[-1] s[-1]), all of which contain the P1-P3 sequence of the enteropeptidase zymogen/activation site. On the basis of its catalytic properties and sites of localization, duodenase has been postulated to be an activator of the enteropeptidase precursor. A tetradecapeptide (LVTQEVSPKIVGGS) having the P9-P5'sequence of the cleavage site of zymogen activation of bovine proenteropeptidase was synthesized, and kinetic parameters of its hydrolysis by duodenase were determined (Km of 87 microM; k[cat] of 1.4 s[-1]; k[cat]/Km of 16000 M[-1] s[-1]). Crystals of duodenase frozen in a stream of liquid nitrogen diffracted synchrotron X-rays to 0.2-nm resolution.

The X-ray structure of the monoclinic crystal form of [D-Hyi2, L-Hyi4] meso-valinomycin.

The conformation and intermolecular association of [D-Hyi2, L-Hyi4] meso-valinomycin [cyclo[-D-Val-D-Hyi-L-Val-L-Hyi-(D-Val-L-Hyi-L-Val-D-+ ++Hyi)2-], C60H102N6O18] in a crystal form obtained from ethanol solution has been determined by x-ray crystallography. Two depsipeptides and one ethanol molecule per asymmetric unit crystallize in space group P2(1) (Z = 4); a = 14.579, b = 39.795, c = 13.928 A, beta = 116.90, Rl = 0.0757. The molecular conformation is very similar to that observed in the trigonal P3(2) crystal form obtained from acetone solution [V. Z. Pletnev et al. (1991) Biopolymers, Vol. 31, pp. 409-415]. Both independent molecules in the crystal adopt a similar distorted bracelet structure with a sterically inaccessible, partially formed, ion-binding center that is stabilized by six 4-->1 type H bonds. The observed conformation accounts for the inability of the molecule to complex ions. Close examination of the three crystallographically independent molecules reveals that differences in the backbone conformation associated with solvent interaction are significantly larger than those associated with hydrophobic van der Waals interactions of crystal packing.

The crystal and molecular structure of a valinomycin analogue cyclo[(D-Val-L-Lac-L-Ala-D-Hyi)2(D-Val-L-Lac-L-Val-D-Hyi)]. H2O(C50H82N6 O18.H2O).

The crystal and molecular structure of the valinomycin analogue, cyclo[(D-Val-L-Lac-L-Ala-D-Hyi)2(D-Val-L-Lac-L-Val-D-Hyi)] has been solved by x-ray direct methods using the "Shake and Bake" procedure. The crystals, grown from a mixture of octane/CH2Cl2, belong to space group P2(1) (Z = 4) with cell parameters a = 10.29, b = 32.08, c = 18.73 A, beta = 97.05 degrees, and contain two molecules per asymmetric unit. After anisotropic refinement the standard reliability factor was Rl = 0.058. The conformations of both independent molecules is similar to that observed for isoleucinomycin, cyclo[-(D-Ile-L-Lac-L-Ile-D-Hyi)3] [V. Z. Pletnev et al. (1980) Biopolymers, Vol. 19, pp. 1517-1534]. The structure has an asymmetric conformation stabilized by six intramolecular H bonds, five bonds being of the 4-->1 type and one bond being of the 5-->1 type. One water molecule is caged in the internal cavity of each cyclodepsipeptide. This conformation could represent an intermediate state between free and complexed forms of valinomycin.

Structure and function of steroid dehydrogenases involved in hypertension, fertility, and cancer.

Short-chain dehydrogenase reductase (SDR) enzymes influence mammalian reproduction, hypertension, neoplasia, and digestion. The three-dimensional structures of two members of the SDR family reveal the position of the conserved catalytic triad, a possible mechanism of keto-hydroxyl interconversion, the molecular mechanism of inhibition, and the basis for selectivity. Glycyrrhizic acid, the active ingredient in licorice, and its metabolite carbenoxolone are potent inhibitors of bacterial 3 alpha, 20 beta-hydroxysteroid dehydrogenase (3 alpha, 20 beta-HSD). The three-dimensional structure of the 3 alpha,20 beta-HSD carbenoxolone complex unequivocally verifies the postulated active site of the enzyme, shows that inhibition is a result of direct competition with the substrate for binding, and provides a plausible model for the mechanism of inhibition of 11 beta-hydroxysteroid dehydrogenase and 15-hydroxyprostaglandin dehydrogenase by carbenoxolone. The structure of human 17 beta-hydroxysteroid dehydrogenase type 1 (17 beta-HSD) suggests the details of binding of estrone and 17 beta-estradiol in the active site of the enzyme and the possible roles of various amino acids in the catalytic cleft. The SDR family includes over 50 proteins from human, mammalian, insect, and bacterial sources. Only five residues are conserved in all members of the family, including the YXXXK sequence. X-ray crystal structures of five members of the family have been completed. When the alpha-carbon backbone of the cofactor binding domains of the five structures are superimposed, the conserved residues are at the core of the structure and in the cofactor binding domain, but not in the substrate binding pocket.