RuvBL1 is an evolutionarily highly conserved eukaryotic protein belonging to the AAA ؉ -family of ATPases (ATPase associated with diverse cellular activities). It plays important roles in essential signaling pathways such as the c-Myc and Wnt pathways in chromatin remodeling, transcriptional and developmental regulation, and DNA repair and apoptosis. Herein we present the three-dimensional structure of the selenomethionine variant of human RuvBL1 refined using diffraction data to 2.2 Å of resolution. The crystal structure of the hexamer is formed of ADP-bound RuvBL1 monomers. The monomers contain three domains, of which the first and the third are involved in ATP binding and hydrolysis. Although it has been shown that ATPase activity of RuvBL1 is needed for several in vivo functions, we could only detect a marginal activity with the purified protein. Structural homology and DNA binding studies demonstrate that the second domain, which is unique among AAA ؉ proteins and not present in the bacterial homolog RuvB, is a novel DNA/RNA-binding domain. We were able to demonstrate that RuvBL1 interacted with single-stranded DNA/RNA and double-stranded DNA. The structure of the RuvBL1⅐ADP complex, combined with our biochemical results, suggest that although RuvBL1 has all the structural characteristics of a molecular motor, even of an ATP-driven helicase, one or more as yet undetermined cofactors are needed for its enzymatic activity.RuvBL1 is a ubiquitously expressed protein (1) that plays important roles in chromatin remodeling, transcription, DNA repair, and apoptosis (2, 3). The significant evolutionary conservation of RuvBL1 from yeast to man strongly suggests that it mediates important cellular functions. RuvBL1 was originally identified by several unrelated approaches and is also known as TIP49a 3 (TATA-binding protein-interacting protein) (1, 4), Rvb1p (5), TAP54␣ (TIP60-associated protein) (3), and Pontin52 (1). The RuvBL1 gene is essential for viability in the yeast Saccharomyces cerevisiae (6), Drosophila melanogaster (7), and in Caenorhabditis elegans.4 A number of chromatin-remodeling complexes contain RuvBL1, like INO80 in yeast and human (8), which is involved in transcription and DNA repair (2), and p400 in animal cells (9). It was demonstrated that yeast Rvb1p is required for the catalytic activity of the INO80 chromatin-remodeling complex (8). RuvBL1 is also an essential component of the human histone acetylase/chromatin-remodeling complex TIP60 (3), which consists of at least 14 distinct subunits and displays histone acetylase activity on chromatin, ATPase, DNA helicase, and structural DNA binding activities.Generally, chromatin-remodeling complexes regulate chromatin structure and are critical for DNA-based transactions in the cell (10, 11). Acetylation of nucleosomal histones leads to relaxation of chromatin structure, thus enabling various transcription factors to gain access to chromatin and to interact with DNA (12, 13). As part of a chromatin-remodeling complex, yeast Rvb1p regulates the transcri...

The crystal structures of the human androgen receptor (hAR) and human progesterone receptor ligandbinding domains in complex with the same ligand metribolone (R1881) have been determined. Both threedimensional structures show the typical nuclear receptor fold. The change of two residues in the ligandbinding pocket between the human progesterone receptor and hAR is most likely the source for the specificity of R1881 to the hAR. The structural implications of the 14 known mutations in the ligand-binding pocket of the hAR ligand-binding domains associated with either prostate cancer or the partial or complete androgen receptor insensitivity syndrome were analyzed. The effects of most of these mutants could be explained on the basis of the crystal structure. Androgen (AR)1 and progesterone receptors (PR) are members of the superfamily of nuclear receptors that includes the steroid receptors, among others, as well as the vitamin D, thyroid, retinoic acid receptors, and the so-called orphan receptors. In addition, AR and PR are members of a group of four closely related steroid receptors including the mineralocorticoid receptor and the glucocorticoid receptor recognizing the same hormone response element. In general, steroid receptors are comprised of five to six domains and act as ligand-activated transcription factors that control the expression of specific genes. To date, no experimentally determined three-dimensional structure is available for a complete receptor. During the past few years, x-ray structures have been published for two of the domains, the DNA-binding domain as well as for a number of ligand-binding domains (LBD) including LBD⅐ligand complexes of the estrogen receptor ␣ and ␤, the PR, the vitamin D receptor, the retinoic acid receptors (X,RXR; acid, RAR), the thyroid hormone receptor, and the peroxisome proliferatoractivated receptors (1-13). Despite the low sequence homology of as low as 20% between the LBDs of different nuclear receptor families, all these proteins share a similar fold. They are comprised of up to 12 helices and a small ␤-sheet arranged in a so-called ␣-helical sandwich, a kind of fold that up to now has only been observed for the LBDs of nuclear receptors. Depending on the nature of the bound ligand, agonist, or antagonist, the carboxyl-terminal helix H12 is found in either one of two orientations. In the agonist-bound conformation, helix H12 serves as a "lid" to close the ligand-binding pocket (LBP), whereas in the antagonist-bound conformation helix H12 is positioned in a different orientation thus opening the entrance to the LBP.Androgens and their receptors play an important role in male physiology and pathology.

Structure-based drug design has often been restricted by the rather static picture of protein–ligand complexes presented by crystal structures, despite the widely accepted importance of protein flexibility in biomolecular recognition. Here we report a detailed experimental and computational study of the drug target, human heat shock protein 90, to explore the contribution of protein dynamics to the binding thermodynamics and kinetics of drug-like compounds. We observe that their binding properties depend on whether the protein has a loop or a helical conformation in the binding site of the ligand-bound state. Compounds bound to the helical conformation display slow association and dissociation rates, high-affinity and high cellular efficacy, and predominantly entropically driven binding. An important entropic contribution comes from the greater flexibility of the helical relative to the loop conformation in the ligand-bound state. This unusual mechanism suggests increasing target flexibility in the bound state by ligand design as a new strategy for drug discovery.

The crystal structure at 2.7 A resolution of the normal human c-H-ras oncogene protein lacking a flexible carboxyl-terminal 18 residue reveals that the protein consists of a six-stranded beta sheet, four alpha helices, and nine connecting loops. Four loops are involved in interactions with bound guanosine diphosphate: one with the phosphates, another with the ribose, and two with the guanine base. Most of the transforming proteins (in vivo and in vitro) have single amino acid substitutions at one of a few key positions in three of these four loops plus one additional loop. The biological functions of the remaining five loops and other exposed regions are at present unknown. However, one loop corresponds to the binding site for a neutralizing monoclonal antibody and another to a putative "effector region"; mutations in the latter region do not alter guanine nucleotide binding or guanosine triphosphatase activity but they do reduce the transforming activity of activated proteins. The data provide a structural basis for understanding the known biochemical properties of normal as well as activated ras oncogene proteins and indicate additional regions in the molecule that may possibly participate in other cellular functions.

Sulfate reduction is one of the earliest types of energy metabolism used by ancestral organisms to sustain life. Despite extensive studies, many questions remain about the way respiratory sulfate reduction is associated with energy conservation. A crucial enzyme in this process is the dissimilatory sulfite reductase (dSiR), which contains a unique siroheme-[4Fe4S] coupled cofactor. Here, we report the structure of desulfoviridin from Desulfovibrio vulgaris, in which the dSiR DsrAB (sulfite reductase) subunits are bound to the DsrC protein. with its conserved C-terminal cysteine reaching the distal side of the siroheme. We propose a novel mechanism for the process of sulfite reduction involving DsrAB, DsrC, and the DsrMKJOP membrane complex (a membrane complex with putative disulfide/thiol reductase activity), in which two of the six electrons for reduction of sulfite derive from the membrane quinone pool. These results show that DsrC is involved in sulfite reduction, which changes the mechanism of sulfate respiration. This has important implications for models used to date ancient sulfur metabolism based on sulfur isotope fractionations.The dissimilatory reduction of sulfur compounds is one of the earliest energy metabolisms detected on earth, at ϳ3.5 billion years ago (1, 2). At the end of the Archean (ϳ2.7 billion years ago), the advent of oxygenic photosynthesis led to a gradual increase in the levels of atmospheric oxygen, which in turn caused an increasing flux of sulfate to the oceans from weathering of sulfide minerals on land (3). As a consequence of this process, reduction of sulfate became a dominant biological process in the oceans, resulting in sulfidic anoxic conditions from about 2.5 to 0.6 billion years ago (3, 4). During this extended period, sulfate-reducing prokaryotes were main players in marine habitats where most evolutionary processes were taking place. Today, these organisms are still major contributors to the biological carbon and sulfur cycles, and their activities have important environmental and economic consequences.A key enzyme in sulfur-based energy metabolism is the dissimilatory sulfite reductase (dSiR), 3 which is present in organisms that reduce sulfate, sulfite, and other sulfur compounds. This enzyme is also found in some phototrophic and chemotrophic sulfur oxidizers, where it is proposed to operate in the reverse direction (reverse sulfite reductase, rSiR). The dSiR is minimally composed of two subunits, DsrA and DsrB, in an ϳ200-kDa ␣ 2 ␤ 2 arrangement. The dsrA and dsrB genes are paralogous and most likely arose from a very early gene duplication event that preceded the separation of the archaea and bacteria domains (5-8), in agreement with a very early onset of biological sulfite reduction. The dSiR belongs to a family of proteins that also include the assimilatory sulfite (aSiR) and nitrite (aNiR) reductases, the monomeric low molecular mass aSiRs, and other dSiRs like asrC and Fsr (9 -11). This family has in common a characteristic cofactor assembly that includes an iro...

No abstract

No abstract

The first crystal structure of a native di-iron center in an iron-storage protein (bacterio)ferritin is reported. The protein, isolated from the anaerobic bacterium Desulfovibrio desulfuricans, has the unique property of having Fe-coproporphyrin III as its heme cofactor. The three-dimensional structure of this bacterioferritin was determined in three distinct catalytic/redox states by X-ray crystallography (at 1.95, 2.05 and 2.35 A resolution), corresponding to different intermediates of the di-iron ferroxidase site. Conformational changes associated with these intermediates support the idea of a route for iron entry into the protein shell through a pore that passes through the di-iron center. Molecular surface and electrostatic potential calculations also suggest the presence of another ion channel, distant from the channels at the three- and four-fold axes proposed as points of entry for the iron atoms.