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A member of the novel protein kinase C (PKC) subfamily, PKCθ, is an essential component of the T cell synapse and is required for optimal T cell activation and interleukin-2 production. Selective involvement of PKCθ in TCR signaling makes this enzyme an attractive therapeutic target in T cell-mediated disease processes. In this report we describe the crystal structure of the catalytic domain of PKCθ at 2.0-A resolution. Human recombinant PKCθ kinase domain was expressed in bacteria as catalytically active phosphorylated enzyme and co-crystallized with its subnanomolar, ATP site inhibitor staurosporine. The structure follows the classic bilobal kinase fold and shows the enzyme in its active conformation and phosphorylated state. Inhibitory interactions between conserved features of staurosporine and the ATP-binding cleft are accompanied by closing of the glycine-rich loop, which also maintains an inhibitory arrangement by blocking the phosphate recognition subsite. The two major phosphorylation sites, Thr-538 in the activation loop and Ser-695 in the hydrophobic motif, are both occupied in the structure, playing key roles in stabilizing active conformation of the enzyme and indicative of PKCθ autocatalytic phosphorylation and activation during bacterial expression. The PKCθ-staurosporine complex represents the first kinase domain crystal structure of any PKC isotypes to be determined and as such should provide valuable insight into PKC specificity and into rational drug design strategies for PKCθ selective leads.

The family of calcium binding proteins called KChIPs associates with Kv4 family K + channels and modulates their biophysical properties. Here, using mutagenesis and X-ray crystallography, we explore the interaction between Kv4 subunits and KChIP1. Two regions in the Kv4.2 N terminus, residues 7–11 and 71–90, are necessary for KChIP1 modulation and interaction with Kv4.2. When inserted into the Kv1.2 N terminus, residues 71–90 of Kv4.2 are also sufficient to confer association with KChIP1. To provide a structural framework for these data, we solved the crystal structures of Kv4.3N and KChIP1 individually. Taken together with the mutagenesis data, the individual structures suggest that that the Kv4 N terminus is required for stable association with KChIP1, perhaps through a hydrophobic surface interaction, and that residues 71–90 in Kv4 subunits form a contact loop that mediates the specific association of KChIPs with Kv4 subunits.

The two known estrogen receptors, ER alpha and ER beta, are hormone inducible transcription factors that have distinct roles in regulating cell proliferation and differentiation. The natural ligand, 17 beta-estradiol (E2), binds with high affinity to both ER alpha and ER beta. However, a close analogue, 16 alpha-iodo-17 beta-estradiol (16 alpha IE2) showed about 10-fold selectivity for ER alpha over ER beta. From X-ray studies, it has been shown that the ligand-binding domains (LBD) of the two receptors are strikingly similar, and that only two changes fall within the binding cavity (ER alpha Leu384 to ER beta Met336, and ER alpha Met421 to ER beta Ile373). To understand the molecular basis for the ER alpha selectivity of 16 alpha IE2, mutants and chimeras of ER alpha and ER beta were generated, and ligand-binding and transactivation functions were studied. The ER alpha Leu384 Met mutant behaved like ER alpha WT in the presence of 16 alpha IE2; whereas the profile of the ER alpha Met421 Ile mutant was similar to that of ER beta WT. The ER beta mutant Ile373 Met behaved like ER alpha with 16 alpha IE2. The results clearly demonstrate the role of ER alpha Met421 in the ER alpha selectivity of 16 alpha IE2.

A total synthesis of the ergot alkaloid paliclavine ( 20 ), in optically active form is described. The synthesis scheme is based on the intramolecular dipolar cycloaddition reaction of a nitrile oxide to a neighboring olefinic appendage bearing an allylic asymmetric center. The extent of diastereofacial selection in the intramolecular nitrile oxide cycloaddition (INOC) reaction was found to be marginal. A single-crystal X-ray analysis has established the complete stereostructure of the isoxazoline 15 prepared from the “major” INOC product. The dependence of the reduction stereochemistry of the isoxazolinium salt 15a on the nature of the reducing agent is discussed.

The intramolecular nitrile oxide cycloaddition reaction has been examined as a method for preparing the hydroazulene ring system.

The reaction of glycosyl acetates with allyltrimethylsilane in the presence of zinc bromide has been investigated as a new method for C-glycoside construction.