Direct mapping of the interface between parathyroid hormone (PTH) and its receptor (hPTH1-Rc Parathyroid hormone (PTH)1 is the major regulator of calcium levels in blood and plays a role in the regulation of bone remodeling (1). Given intermittently, PTH displays anabolic activity in bone and, therefore, has considerable therapeutic potential (2). PTH and PTH-related protein exert their actions via a seven-transmembrane (TM) domain-containing receptor (PTH1-Rc) (3) belonging to a subfamily of related G proteincoupled receptors (4 -11). The PTH1-Rc is coupled to both adenylyl cyclase/cyclic AMP and phospholipase C/inositol 1,4,5-trisphosphate/cytosolic calcium intracellular signaling pathways (12)(13)(14)(15).Understanding the molecular mechanism of ligand recognition and signal transduction by the PTH1-Rc may identify new directions for the design of novel hormone analogs for the treatment of diseases such as osteoporosis, hypercalcemia of malignancy and hyperparathyroidism (16). In order to directly identify the structural elements involved in PTH-PTH1-Rc interactions, we employed a photoaffinity scanning approach (17). The generation of covalently linked ligand-receptor conjugates and the identification of the cross-linked domains allows mapping of the interface between hormone and receptor. Photoaffinity cross-linking has been successfully applied in defining interactions between small peptides, such as substance P (18 -20), cholecystokinin (21), and vasopressin (22), and their receptors. Recently, we used this general approach to identify directly the interaction between position 13 of PTH and a 17-amino acid domain (residues 173-189) of the hPTH1-Rc (17).We now report the evaluation of a series of photoreactive analogs obtained by a "p-benzoylphenylalanine (Bpa) scan" of the principal receptor activation domain (residues 1-6) of 34)), maintained full potency and led to the identification of a second "contact domain" between PTH and hPTH1-Rc. This information allows us to create, for the first time, a model describing interactions of hPTH-(1-34) with its receptor based on direct identification of the interacting regions. EXPERIMENTAL PROCEDURESMaterials-Boc-protected amino acids, N-hydroxybenzotriazole, N,NЈ-dicyclohexylcarbodiimide, and p-methylbenzydrylamine resin were purchased from Applied Biosystems (Foster City, CA). Boc-(3-iodo)tyrosine ] was from Peninsula Laboratories (Belmont, CA). B&J brand dichloromethane, N-methylpyrrolidone, and acetonitrile were obtained from Baxter (McGraw Park, IL). IODOGEN ® and 2-(2Ј-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine (BNPS-skatole) were purchased from Pierce. Cyanogen bromide was from Aldrich. Na

The phosphorylation of IkappaB by the IKK complex targets it for degradation and releases NF-kappaB for translocation into the nucleus to initiate the inflammatory response, cell proliferation, or cell differentiation. The IKK complex is composed of the catalytic IKKalpha/beta kinases and a regulatory protein, NF-kappaB essential modulator (NEMO; IKKgamma). NEMO associates with the unphosphorylated IKK kinase C termini and activates the IKK complex's catalytic activity. However, detailed structural information about the NEMO/IKK interaction is lacking. In this study, we have identified the minimal requirements for NEMO and IKK kinase association using a variety of biophysical techniques and have solved two crystal structures of the minimal NEMO/IKK kinase associating domains. We demonstrate that the NEMO core domain is a dimer that binds two IKK fragments and identify energetic hot spots that can be exploited to inhibit IKK complex formation with a therapeutic agent.

Cholera is an acute intestinal infection caused by the bacterium Vibrio cholerae. In order for V. cholerae to cause disease, it must produce two virulence factors, the toxin-coregulated pilus (TCP) and cholera toxin (CT), whose expression is controlled by a transcriptional cascade culminating with the expression of the AraCfamily regulator, ToxT. We have solved the 1.9 Å resolution crystal structure of ToxT, which reveals folds in the N-and C-terminal domains that share a number of features in common with AraC, MarA, and Rob as well as the unexpected presence of a buried 16-carbon fatty acid, cis-palmitoleate. The finding that cis-palmitoleic acid reduces TCP and CT expression in V. cholerae and prevents ToxT from binding to DNA in vitro provides a direct link between the host environment of V. cholerae and regulation of virulence gene expression.AraC | crystal structure | pathogenesis | oleic acid | palmitoleic acid

The bimolecular complex of the C-terminal octapeptide of cholecystokinin, CCK-8, with the N-terminus of the CCK(A)-receptor, CCK(A)-R(1-47), has been structurally characterized by high-resolution NMR and computational refinement. The conformation of CCK(A)-R(1-47), within the lipid environment used for the spectroscopic studies, consists of a well-defined alpha-helix (residues 3-9) followed by a beta-sheet stabilized by a disulfide linkage between C18 and C29, leading to the first transmembrane alpha-helix (TM1). Titration of CCK(A)-R(1-47) with CCK-8 specifically affects the NMR signals of W39 of the receptor, in a saturable fashion. This association is specific for CCK-8; no association was observed upon titration of CCK(A)-R(1-47) with other peptide hormones. The ligand/receptor complex was characterized by intermolecular NOEs between Tyr(27) and Met(28) of CCK-8 and W39 of CCK(A)-R(1-47). These findings suggest that CCK-8 binds to CCK(A) with the C-terminus within the seven-helical bundle and the N-terminus of the ligand, projecting out between TM1 and TM7, forming specific interactions with the N-terminus of the CCK(A) receptor. This mode of ligand binding, consistent with published mutagenesis studies, requires variation of the interpretation of recent findings from photoaffinity cross-linking studies.