Structure-guided disruption of the pseudopilus tip complex inhibits the Type II secretion in Pseudomonas aeruginosa.

Pseudomonas aeruginosa utilizes the Type II secretion system (T2SS) to translocate a wide range of large, structured protein virulence factors through the periplasm to the extracellular environment for infection. In the T2SS, five pseudopilins assemble into the pseudopilus that acts as a piston to extrude exoproteins out of cells. Through structure determination of the pseudopilin complexes of XcpVWX and XcpVW and function analysis, we have confirmed that two minor pseudopilins, XcpV and XcpW, constitute a core complex indispensable to the pseudopilus tip. The absence of either XcpV or -W resulted in the non-functional T2SS. Our small-angle X-ray scattering experiment for the first time revealed the architecture of the entire pseudopilus tip and established the working model. Based on the interaction interface of complexes, we have developed inhibitory peptides. The structure-based peptides not only disrupted of the XcpVW core complex and the entire pseudopilus tip in vitro but also inhibited the T2SS in vivo. More importantly, these peptides effectively reduced the virulence of P. aeruginosa towards Caenorhabditis elegans.

Scaffoldin conformation and dynamics revealed by a ternary complex from the Clostridium thermocellum cellulosome.

Cellulosomes are multienzyme complexes responsible for efficient degradation of plant cell wall polysaccharides. The nonenzymatic scaffoldin subunit provides a platform for cellulolytic enzyme binding that enhances the overall activity of the bound enzymes. Understanding the unique quaternary structural elements responsible for the enzymatic synergy of the cellulosome is hindered by the large size and inherent flexibility of these multiprotein complexes. Herein, we have used x-ray crystallography and small angle x-ray scattering to structurally characterize a ternary protein complex from the Clostridium thermocellum cellulosome that comprises a C-terminal trimodular fragment of the CipA scaffoldin bound to the SdbA type II cohesin module and the type I dockerin module from the Cel9D glycoside hydrolase. This complex represents the largest fragment of the cellulosome solved by x-ray crystallography to date and reveals two rigid domains formed by the type I cohesin·dockerin complex and by the X module-type II cohesin·dockerin complex, which are separated by a 13-residue linker in an extended conformation. The type I dockerin modules of the four structural models found in the asymmetric unit are in an alternate orientation to that previously observed that provides further direct support for the dual mode of binding. Conserved intermolecular contacts between symmetry-related complexes were also observed and may play a role in higher order cellulosome structure. SAXS analysis of the ternary complex revealed that the 13-residue intermodular linker of the scaffoldin subunit is highly dynamic in solution. These studies provide fundamental insights into modular positioning, linker flexibility, and higher order organization of the cellulosome.

High-resolution structure of AKR1a4 in the apo form and its interaction with ligands.

Aldo-keto reductase 1a4 (AKR1a4; EC 1.1.1.2) is the mouse orthologue of human aldehyde reductase (AKR1a1), the founding member of the AKR family. As an NADPH-dependent enzyme, AKR1a4 catalyses the conversion of D-glucuronate to L-gulonate. AKR1a4 is involved in ascorbate biosynthesis in mice, but has also recently been found to interact with SMAR1, providing a novel mechanism of ROS regulation by ATM. Here, the crystal structure of AKR1a4 in its apo form at 1.64 Šresolution as well as the characterization of the binding of AKR1a4 to NADPH and P44, a peptide derived from SMAR1, is presented.

8-oxoguanine DNA glycosylases: one lesion, three subfamilies.

Amongst the four bases that form DNA, guanine is the most susceptible to oxidation, and its oxidation product, 7,8-dihydro-8-oxoguanine (8-oxoG) is the most prevalent base lesion found in DNA. Fortunately, throughout evolution cells have developed repair mechanisms, such as the 8-oxoguanine DNA glycosylases (OGG), which recognize and excise 8-oxoG from DNA thereby preventing the accumulation of deleterious mutations. OGG are divided into three subfamilies, OGG1, OGG2 and AGOG, which are all involved in the base excision repair (BER) pathway. The published structures of OGG1 and AGOG, as well as the recent availability of OGG2 structures in both apo- and liganded forms, provide an excellent opportunity to compare the structural and functional properties of the three OGG subfamilies. Among the observed differences, the three-dimensional fold varies considerably between OGG1 and OGG2 members, as the latter lack the A-domain involved in 8-oxoG binding. In addition, all three OGG subfamilies bind 8-oxoG in a different manner even though the crucial interaction between the enzyme and the protonated N7 of 8-oxoG is conserved. Finally, the three OGG subfamilies differ with respect to DNA binding properties, helix-hairpin-helix motifs, and specificity for the opposite base.

Purification, crystallization and preliminary X-ray diffraction of a disulfide cross-linked complex between bovine poly(A) polymerase and a chemically modified 15-mer oligo(A) RNA.

Poly(A) polymerase (PAP) synthesizes the polyadenine tail at the 3'-end of messenger RNA. A disulfide cross-linking strategy was implemented to obtain a complex between bovine PAP (bPAP) and a 15-mer oligo(A). All seven endogenous cysteines were mutated to eliminate nonspecific cross-linked complexes. A cysteine residue was introduced at several different positions and A152C was found to achieve maximum specific cross-linking efficiency. The resulting bPAP construct was active and, when mixed with a chemically modified RNA, yielded crystals of a bPAP-RNA complex. The crystals, which belonged to space group P2 and harbored two protein-RNA complexes per asymmetric unit, diffracted X-rays to 2.25 Šresolution.

Crystal structures of two archaeal 8-oxoguanine DNA glycosylases provide structural insight into guanine/8-oxoguanine distinction.

Among the four DNA bases, guanine is particularly vulnerable to oxidative damage and the most common oxidative product, 7,8-dihydro-8-oxoguanine (8-oxoG), is the most prevalent lesion observed in DNA molecules. Fortunately, 8-oxoG is recognized and excised by the 8-oxoguanine DNA glycosylase (Ogg) of the base excision repair pathway. Ogg enzymes are divided into three separate families, namely, Ogg1, Ogg2, and archaeal GO glycosylase (AGOG). To date, structures of members of both Ogg1 and AGOG families are known but no structural information is available for members of Ogg2. Here we describe the first crystal structures of two archaeal Ogg2: Methanocaldococcus janischii Ogg and Sulfolobus solfataricus Ogg. A structural comparison with OGG1 and AGOG suggested that the C-terminal lysine of Ogg2 may play a key role in discriminating between guanine and 8-oxoG. This prediction was substantiated by measuring the glycosylase/lyase activity of a C-terminal deletion mutant of MjaOgg.

Progesterone metabolism in adipose cells.

The aim of the present study was to investigate pathways of progesterone metabolism in human adipose cells. Adipose tissue samples from the omental (OM) and subcutaneous (SC) fat compartments were surgically obtained in women. In isolated mature adipocytes, progesterone was converted to 20alpha-hydroxyprogesterone as the main metabolite, most likely through the activity of aldo-keto reductases 1C1, 2 and 3 (20alpha-HSD, 3alpha-HSD type 3 and 17beta-HSD type 5, respectively). In cultured preadipocytes, progesterone was converted to several metabolites identified using bidimensional thin layer chromatography, with or without the dual inhibitor of 5alpha-reductase type 1 and 2 (17beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5alpha-androstan-3-one (4-MA)). Major metabolites identified in OM and SC preadipocytes which were incubated for 24h with (14)C-labelled progesterone were 20alpha-hydroxyprogesterone, 5alpha-pregnane-3alpha/beta-ol-20-one, 5alpha- and 5beta-pregnanedione, 5alpha- and 5beta-pregnane-20alpha-ol-3-one, 5alpha-pregnane-3alpha/beta-ol-20-one and 5beta-pregnane-3alpha/beta-20alpha-diol. Induction of preadipocyte differentiation increased expression levels of AKR1C1 and modified the pattern of progesterone metabolism substantially, leaving 20alpha-hydroxyprogesterone as the main metabolite generated. On the other hand, progesterone itself showed no consistent effect on adipocyte differentiation. In conclusion, preadipocytes and lipid-storing, mature adipocytes efficiently generate progesterone metabolites in women, which is consistent with rather modest effects progesterone on abdominal fat cell differentiation.

Crystal structures of human Delta4-3-ketosteroid 5beta-reductase (AKR1D1) reveal the presence of an alternative binding site responsible for substrate inhibition.

The 5beta-reductases (AKR1D1-3) are unique enzymes able to catalyze efficiently and in a stereospecific manner the 5beta-reduction of the C4-C5 double bond found in Delta4-3-ketosteroids, including steroid hormones and bile acids precursors such as 7alpha-hydroxy-4-cholesten-3-one and 7alpha,12alpha-dihydroxy-4-cholesten-3-one. In order to elucidate the binding mode and substrate specificity in detail, biochemical and structural studies on human 5beta-reductase (h5beta-red; AKR1D1) have been recently undertaken. The crystal structure of a h5beta-red binary complex provides a complete picture of the NADPH-enzyme interactions involving the flexible loop B, which contributes to the maintenance of the cofactor in its binding site by acting as a "safety belt". Structural comparison with binary complexes of AKR1C enzymes, specifically the human type 3 3alpha-hydroxysteroid dehydrogenase (AKR1C2) and the mouse 17alpha-hydroxysteroid dehydrogenase (AKR1C21), also revealed particularities in loop B positioning that make the steroid-binding cavity of h5beta-red substantially larger than those of the two other enzymes. Kinetic characterization of the purified recombinant h5beta-red has shown that this enzyme exerts a strong activity toward progesterone (Prog) and androstenedione (Delta4) but is rapidly inhibited by these substrates once their concentrations reach 2-times their K(m) value. A crystal structure of the h5beta-red in ternary complex with NADPH and Delta4 has revealed that the large steroid-binding site of this enzyme also contains a subsite in which the Delta4 molecule is found. When bound in this subsite, Delta4 completely impedes the passage of another substrate molecule toward the catalytic site. The importance of this alternative binding site for the inhibition of h5beta-red was finally proven by site-directed mutagenesis, which demonstrated that the replacement of one of the residues delineating this site (Val(309)) by a phenylalanine completely abolishes the substrate inhibition. The results of this report provide structural insights into the substrate inhibition of h5beta-red by C19- and C21-steroids.

The crystal structure of human Delta4-3-ketosteroid 5beta-reductase defines the functional role of the residues of the catalytic tetrad in the steroid double bond reduction mechanism.

The 5beta-reductases (AKR1D1-3) are unique enzymes able to catalyze efficiently and in a stereospecific manner the 5beta-reduction of the C4-C5 double bond found into Delta4-3-ketosteroids, including steroid hormones and bile acids. Multiple-sequence alignments and mutagenic studies have already identified one of the residues presumably located at their active site, Glu 120, as the major molecular determinant for the unique activity displayed by 5beta-reductases. To define the exact role played by this glutamate in the catalytic activity of these enzymes, biochemical and structural studies on human 5beta-reductase (h5beta-red) have been undertaken. The crystal structure of h5beta-red in a ternary complex with NADP (+) and 5beta-dihydroprogesterone (5beta-DHP), the product of the 5beta-reduction of progesterone (Prog), revealed that Glu 120 does not interact directly with the other catalytic residues, as previously hypothesized, thus suggesting that this residue is not directly involved in catalysis but could instead be important for the proper positioning of the steroid substrate in the catalytic site. On the basis of our structural results, we thus propose a realistic scheme for the catalytic mechanism of the C4-C5 double bond reduction. We also propose that bile acid precursors such as 7alpha-hydroxy-4-cholesten-3-one and 7alpha,12alpha-dihydroxy-4-cholesten-3-one, when bound to the active site of h5beta-red, can establish supplementary contacts with Tyr 26 and Tyr 132, two residues delineating the steroid-binding cavity. These additional contacts very likely account for the higher activity of h5beta-red toward the bile acid intermediates versus steroid hormones. Finally, in light of the structural data now available, we attempt to interpret the likely consequences of mutations already identified in the gene encoding the h5beta-red enzyme which lead to a reduction of its enzymatic activity and which can progress to severe liver function failure.

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.

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.

Characterization of 17alpha-hydroxysteroid dehydrogenase activity (17alpha-HSD) and its involvement in the biosynthesis of epitestosterone.

BACKGROUND: Epi-testosterone (epiT) is the 17alpha-epimer of testosterone. It has been found at similar level as testosterone in human biological fluids. This steroid has thus been used as a natural internal standard for assessing testosterone abuse in sports. EpiT has been also shown to accumulate in mammary cyst fluid and in human prostate. It was found to possess antiandrogenic activity as well as neuroprotective effects. So far, the exact pathway leading to the formation of epiT has not been elucidated. RESULTS: In this report, we describe the isolation and characterization of the enzyme 17alpha-hydroxysteroid dehydrogenase. The name is given according to its most potent activity. Using cells stably expressing the enzyme, we show that 17alpha-HSD catalyzes efficienty the transformation of 4-androstenedione (4-dione), dehydroepiandrosterone (DHEA), 5alpha-androstane-3,17-dione (5alpha-dione) and androsterone (ADT) into their corresponding 17alpha-hydroxy-steroids : epiT, 5-androstene-3beta,17alpha-diol (epi5diol), 5alpha-androstane-17alpha-ol-3-one (epiDHT) and 5alpha-androstane-3alpha,17alpha-diol (epi3alpha-diol), respectively. Similar to other members of the aldo-keto reductase family that possess the ability to reduce the keto-group into hydroxyl-group at different position on the steroid nucleus, 17alpha-HSD could also catalyze the transformation of DHT, 5alpha-dione, and 5alpha-pregnane-3,20-dione (DHP) into 3alpha-diol, ADT and 5alpha-pregnane-3alpha-ol-20-one (allopregnanolone) through its less potent 3alpha-HSD activity. We also have over-expressed the 17alpha-HSD in Escherichia coli and have purified it by affinity chromatography. The purified enzyme exhibits the same catalytic properties that have been observed with cultured HEK-293 stably transfected cells. Using quantitative Realtime-PCR to study tissue distribution of this enzyme in the mouse, we observed that it is expressed at very high levels in the kidney. CONCLUSION: The present study permits to clarify the biosynthesis pathway of epiT. It also offers the opportunity to study gene regulation and function of this enzyme. Further study in human will allow a better comprehension about the use of epiT in drug abuse testing; it will also help to clarify the importance of its accumulation in breast cyst fluid and prostate, as well as its potential role as natural antiandrogen.

Growth of Diffraction-Quality Protein Crystals Using a Harvestable Microfluidic Device.

Protein crystallization is the major bottleneck in the entire process of protein crystallography, and obtaining diffraction-quality crystals can be unpredictable and sometimes exceptionally difficult, requiring many rounds of high-throughput screening. Recently, a more time- and cost-saving strategy to use the commercially available microfluidic devices called Crystal Formers has emerged. Herein we show the application of such a device using a protein from Legionella pneumophila called LidL that is predicted to be involved in the ability to efficiently manipulate host cell trafficking events once internalized by the host cell. After setting up just one 96-channel Crystal Former tray, we were able to obtain a diffraction-quality crystal that diffracted to 2.76 Å. These results show that Crystal Formers can be used to screen and optimize crystals to directly produce crystals for structure determination.

Structural basis of calcineurin activation by calmodulin.

Calcineurin is the only known calmodulin (CaM) activated protein phosphatase, which is involved in the regulation of numerous cellular and developmental processes and in calcium-dependent signal transduction. Although commonly assumed that CaM displaces the autoinhibitory domain (AID) blocking substrate access to its active site, the structural basis underlying activation remains elusive. We have created a fused ternary complex (CBA) by covalently linking three polypeptides: CaM, calcineurin regulatory B subunit (CnB) and calcineurin catalytic A subunit (CnA). CBA catalytic activity is comparable to that of fully activated native calcineurin in the presence of CaM. The crystal structure showed virtually no structural change in the active site and no evidence of CaM despite being covalently linked. The asymmetric unit contains four molecules; two parallel CBA pairs are packed in an antiparallel mode and the large cavities in crystal packing near the calcineurin active site would easily accommodate multiple positions of AID-bound CaM. Intriguingly, the conformation of the ordered segment of AID is not altered by CaM; thus, it is the disordered part of AID, which resumes a regular α-helical conformation upon binding to CaM, which is displaced by CaM for activation. We propose that the structural basis of calcineurin activation by CaM is through displacement of the disordered fragment of AID which otherwise impedes active site access.

Antifreeze protein from freeze-tolerant grass has a beta-roll fold with an irregularly structured ice-binding site.

The grass Lolium perenne produces an ice-binding protein (LpIBP) that helps this perennial tolerate freezing by inhibiting the recrystallization of ice. Ice-binding proteins (IBPs) are also produced by freeze-avoiding organisms to halt the growth of ice and are better known as antifreeze proteins (AFPs). To examine the structural basis for the different roles of these two IBP types, we have solved the first crystal structure of a plant IBP. The 118-residue LpIBP folds as a novel left-handed beta-roll with eight 14- or 15-residue coils and is stabilized by a small hydrophobic core and two internal Asn ladders. The ice-binding site (IBS) is formed by a flat beta-sheet on one surface of the beta-roll. We show that LpIBP binds to both the basal and primary-prism planes of ice, which is the hallmark of hyperactive AFPs. However, the antifreeze activity of LpIBP is less than 10% of that measured for those hyperactive AFPs with convergently evolved beta-solenoid structures. Whereas these hyperactive AFPs have two rows of aligned Thr residues on their IBS, the equivalent arrays in LpIBP are populated by a mixture of Thr, Ser and Val with several side-chain conformations. Substitution of Ser or Val for Thr on the IBS of a hyperactive AFP reduced its antifreeze activity. LpIBP may have evolved an IBS that has low antifreeze activity to avoid damage from rapid ice growth that occurs when temperatures exceed the capacity of AFPs to block ice growth while retaining the ability to inhibit ice recrystallization.

The C-terminal lysine of Ogg2 DNA glycosylases is a major molecular determinant for guanine/8-oxoguanine distinction.

7,8-Dihydro-8-oxoguanine (8-oxoG) is a major oxidative lesion found in DNA. The 8-oxoguanine DNA glycosylases (Ogg) responsible for the removal of 8-oxoG are divided into three families Ogg1, Ogg2 and AGOG. The Ogg2 members are devoid of the recognition loop used by Ogg1 to discriminate between 8-oxoG and guanine and it was unclear until recently how Ogg2 enzymes recognize the oxidized base. We present here the first crystallographic structure of an Ogg2 member, Methanocaldococcus janischii Ogg, in complex with a DNA duplex containing the 8-oxoG lesion. This structure highlights the crucial role of the C-terminal lysine, strictly conserved in Ogg2, in the recognition of 8-oxoG. The structure also reveals that Ogg2 undergoes a conformational change upon DNA binding similar to that observed in Ogg1 glycosylases. Furthermore, this work provides a structural rationale for the lack of opposite base specificity in this family of enzymes.

Structural basis for the lack of opposite base specificity of Clostridium acetobutylicum 8-oxoguanine DNA glycosylase.

7,8-Dihydro-8-oxoguanine (8-oxoG) is the major oxidative product of guanine and the most prevalent base lesion observed in DNA molecules. Because 8-oxoG has the capability to form a Hoogsteen pair with adenine (8-oxoG:A) in addition to a normal Watson-Crick pair with cytosine (8-oxoG:C), this lesion can lead to a G:C-->T:A transversion after replication. However, 8-oxoG is recognized and excised by the 8-oxoguanine DNA glycosylase (Ogg) of the base excision repair pathway. Members of the Ogg1 family usually display a strong preference for a C opposite the lesion. In contrast, the atypical Ogg1 from Clostridium actetobutylicum (CacOgg) can excise 8-oxoG when paired with either one of the four bases, albeit with a preference for C and A. Here we describe the first high-resolution crystal structures of CacOgg in complex with duplex DNA containing the 8-oxoG lesion paired to cytosine and to adenine. A structural comparison with human OGG1 provides a rationale for the lack of opposite base specificity displayed by the bacterial Ogg.

Structural characterization of Clostridium acetobutylicum 8-oxoguanine DNA glycosylase in its apo form and in complex with 8-oxodeoxyguanosine.

DNA is subject to a multitude of oxidative damages generated by oxidizing agents from metabolism and exogenous sources and by ionizing radiation. Guanine is particularly vulnerable to oxidation, and the most common oxidative product 8-oxoguanine (8-oxoG) is the most prevalent lesion observed in DNA molecules. 8-OxoG can form a normal Watson-Crick pair with cytosine (8-oxoG:C), but it can also form a stable Hoogsteen pair with adenine (8-oxoG:A), leading to a G:C-->T:A transversion after replication. Fortunately, 8-oxoG is recognized and excised by either of two DNA glycosylases of the base excision repair pathway: formamidopyrimidine-DNA glycosylase and 8-oxoguanine DNA glycosylase (Ogg). While Clostridium acetobutylicum Ogg (CacOgg) DNA glycosylase can specifically recognize and remove 8-oxoG, it displays little preference for the base opposite the lesion, which is unusual for a member of the Ogg1 family. This work describes the crystal structures of CacOgg in its apo form and in complex with 8-oxo-2'-deoxyguanosine. A structural comparison between the apo form and the liganded form of the enzyme reveals a structural reorganization of the C-terminal domain upon binding of 8-oxoG, similar to that reported for human OGG1. A structural comparison of CacOgg with human OGG1, in complex with 8-oxoG containing DNA, provides a structural rationale for the lack of opposite base specificity displayed by CacOgg.

Structural characterization of the human androgen receptor ligand-binding domain complexed with EM5744, a rationally designed steroidal ligand bearing a bulky chain directed toward helix 12.

Antiandrogens are commonly used to treat androgen-dependent disorders. The currently used drugs unfortunately possess very weak affinity for the human AR (hAR), thus indicating the need to develop new high-affinity steroidal antiandrogens. Our compounds are specially designed to impede repositioning of the mobile carboxyl-terminal helix 12, which blocks the ligand-dependent transactivation function (AF-2) located in the AR ligand-binding domain (ARLBD). Using crystal structures of the hARLBD, we first found that H12 could be directly reached from the ligand-binding pocket (LBP) by a chain positioned on the C18 atom of an androgen steroid nucleus. A set of 5alpha-dihydrotestosterone-derived molecules bearing various C18 chains were thus synthesized and tested for their capacity to bind hAR and act as antagonists. Although most of those having very high affinity for hAR were agonists, several very potent antagonists were obtained, confirming the structural importance of the C18 chain. To understand the role of the C18 chain in their agonistic/antagonistic properties, the structure of the hARLBD complexed with one of these agonists, EM5744, was determined at a 1.65-A resolution. We have identified new interactions involving Gln(738), Met(742), and His(874) that explain both the high affinity of this compound and the inability of its bulky chain to prevent the repositioning of H12. This structural information will be helpful to refine the structure of the chains placed on the C18 atom to obtain efficient H12-directed steroidal antiandrogens.