The crystal structure of the serine protease subtllsln Carbsberg In anhydrous acetonitrile was determined at 2.3 A rolution. It was found to be easentially Identical to the tbree-dimensional structure of the enzyme In water; the differences observed were smaller than those between two lndependentiy determined struchre In aqueous solution. The hydrogen bond system of the catalytic triad is intact in acetonitrile. The majority (99 of 119) of enzyme-bound, structural water molcules have such a great affnity to subillsin that they are not dipld even in anhydrous acetonltrile. Of the 12 enzyme-bound acetonitrile mdsoules, 4 dispiace water molecules and 8 bind where no water had been observed before. One-third of all subtin-bound acetonitrile molecules reside in the active center, occupying the same region (PI, P2, and P3 bnding dtes) as the specfic protein Inhibitor eglin c.The past several years have witnessed the emergence of nonaqueous enzymology (1-5); it has become clear that not only can ordinary enzymes [and antibodies (6-8)] function in organic solvents containing little or no water, but when placed in such an utterly unnatural milieu they exhibit unusual properties, including the ability to catalyze additional reactions, markedly altered specificity, and "molecular memory." To understand the mechanistic basis of these phenomena, it is critical to elucidate protein conformation in organic media and compare it with that in aqueous solution.This task has been achieved in the present work by means of high-resolution x-ray crystallography; remarkably, the threedimensional structure of the serine protease subtilisin Carlsberg in anhydrous acetonitrile has been found to be essentially indistinguishable from that in water. § Moreover, much of the water structure around the protein is identical to that in aqueous medium. In addition, several acetonitrile molecules are uniquely bound to the protein, mapping an interaction surface distinct from that for water. METHODS Cyt. Subtilisin Carlsberg, an alkaline serine protease from Bacillus licheniformis (EC 3.4.21.14) purchased from Sigma (type VIII), was crystallized by batch precipitationtfrom aqueous Na2SO4 as described (9). The long prismatic crystals obtained were divided into two portions. The first was used to determine the enzyme structure in water at a higher resolution than is currently available (10), so that the number and positions of bound water molecules could be established. The second batch of crystals was used for nonaqueous structure analysis. Solvent Inion. Attempts to gradually replace the interstitial water with acetonitrile failed; when the organic solvent content reached aw50%6 (vol/vol), the enzyme crystals invariably fractured. To fortify the crystals, we cross-linked them lightly with glutaraldehyde following the general methodology of . [Cross-linked enzyme

Nuclear receptors (NRs) form a superfamily of ligand-inducible transcription factors composed of several domains. Recent structural studies focused on domain E, which harbors the ligand-binding site and the ligand-dependent transcription activation function AF-2. Structures of single representatives in an increasing number of various complexes as well as new structures of further NRs addressed issues such as discrimination of ligands, superagonism, isotype specificity, and partial agonism. Until today, one unique transcriptionally active form of domain E was determined; however, divergent tertiary structures of apo-forms and transcriptionally inactive forms are known. Thus, recent results link the transformation of NRs upon ligand binding to principles of protein folding. Furthermore, the ensemble of NR structures, including those of DNA-binding domains, provides one of the foundations for the understanding of interactions with transcription intermediary factors up to the characterization of the link between NR complexes and the basal transcriptional machinery at the structural level.

The retinoic acid-related orphan receptor beta (RORbeta) exhibits a highly restricted neuronal-specific expression pattern in brain, retina and pineal gland. So far, neither a natural RORbeta target gene nor a functional ligand have been identified, and the physiological role of the receptor is not well understood. We present the crystal structure of the ligand-binding domain (LBD) of RORbeta containing a bound stearate ligand and complexed with a coactivator peptide. In the crystal, the monomeric LBD adopts the canonical agonist-bound form. The fatty acid ligand-coactivator peptide combined action stabilizes the transcriptionally active conformation. The large ligand-binding pocket is strictly hydrophobic on the AF-2 side and more polar on the beta-sheet side where the carboxylate group of the ligand binds. Site-directed mutagenesis experiments validate the significance of the present structure. Homology modeling of the other isotypes will help to design isotype-selective agonists and antagonists that can be used to characterize the physiological functions of RORs. In addition, our crystallization strategy can be extended to other orphan nuclear receptors, providing a powerful tool to delineate their functions.