Although AKT1 (v-akt murine thymoma viral oncogene homologue 1) kinase is a central member of possibly the most frequently activated proliferation and survival pathway in cancer, mutation of AKT1 has not been widely reported. Here we report the identification of a somatic mutation in human breast, colorectal and ovarian cancers that results in a glutamic acid to lysine substitution at amino acid 17 (E17K) in the lipid-binding pocket of AKT1. Lys 17 alters the electrostatic interactions of the pocket and forms new hydrogen bonds with a phosphoinositide ligand. This mutation activates AKT1 by means of pathological localization to the plasma membrane, stimulates downstream signalling, transforms cells and induces leukaemia in mice. This mechanism indicates a direct role of AKT1 in human cancer, and adds to the known genetic alterations that promote oncogenesis through the phosphatidylinositol-3-OH kinase/AKT pathway. Furthermore, the E17K substitution decreases the sensitivity to an allosteric kinase inhibitor, so this mutation may have important clinical utility for AKT drug development.

Mutations in the obese gene (OB) or in the gene encoding the OB receptor(OB-R) result in obesity, infertility and diabetes in a variety of mouse phenotypes. The demonstration that OB protein (also known as leptin) can normalize body weight in ob/ob mice has generated enormous interest. Most human obesity does not appear to result from a mutant form of leptin: rather, serum leptin concentrations are increased and there is an apparent inability to transport it to the central nervous system (CNS). Injection of leptin into the CNS of overfed rodents resistant to peripheral administration was found to induce biological activity. Consequently, for the leptin to act as a weight-lowering hormone in human obesity, it appears that appropriate concentrations must be present in the CNS. This places a premium on understanding the structure of the hormone in order to design more potent and selective agonists. Here we report the crystal structure at 2.4A resolution of a human mutant OB protein (leptin-E100) that has comparable biological activity to wild type but which crystallizes more readily. The structure reveals a four-helix bundle similar to that of the long-chain helical cytokine family.

The interaction between nuclear receptors and coactivators provides an arena for testing whether protein-protein interactions may be inhibited by small molecule drug candidates. We provide evidence that a short cyclic peptide, containing a copy of the LXXLL nuclear receptor box pentapeptide, binds tightly and selectively to estrogen receptor ␣. Furthermore, as shown by x-ray analysis, the disulfide-bridged nonapeptide, nonhelical in aqueous solutions, is able to adopt a quasihelical conformer while binding to the groove created by ligand attachment to estrogen receptor ␣. An i, i؉3 linked analog, H-Lys-cyclo(D-Cys-Ile-Leu-Cys)-Arg-Leu-Leu-Gln-NH 2 (peptidomimetic estrogen receptor modulator 1), binds with a Ki of 25 nM, significantly better than an i, i؉4 bridged cyclic amide, as predicted by molecular modeling design criteria. The induction of helical character, effective binding, and receptor selectivity exhibited by this peptide analog provide strong support for this strategy. The stabilization of minimalist surface motifs may prove useful for the control of other macromolecular assemblies, especially when an amphiphilic helix is crucial for the strong binding interaction between two proteins. M embers of the nuclear receptor (NR) superfamily, which include the steroid receptors, are ligand-activated transcription factors that regulate a wide variety of physiological and developmental processes (1-3). Upon ligand binding, steroid receptors shed their accompanying heat shock proteins to form homodimers, and bind to their cognate DNA elements within the regulatory regions of steroid responsive genes. Steroid receptor agonists are typically hydrophobic molecules and have been demonstrated to bind to a buried hydrophobic pocket within the carboxyterminal ligand-binding domain (LBD) of the receptor. This results in a conformational shift causing repositioning of helix 12, which allows for recognition of coactivator proteins. Many coactivators contain a short pentapeptide motif, known as a NR box (4), that is responsible for recognition of a hydrophobic groove created on the surface of the LBD in response to repositioning of helix 12 upon agonist binding (5). Steroid receptor antagonists, like agonists, are also hydrophobic molecules and bind within the core of the LBD; however, these ligands do not position helix 12 in the correct conformation that would allow the coactivators to recognize the receptor. A large number of proteins characterized as NR coactivators have been identified, and many appear to contain one or, in some cases, multiple copies of the NR box with the consensus sequence LXXLL. McDonnell and coworkers (6, 7) have noted that peptide sequences that mimic this NR interaction motif could function as ER antagonists in cell based models when overexpressed as a component of a fusion protein. Detailed analysis of the interactions between the receptor and coactivators has revealed new possible points of intervention (8). Such targets have recently been proposed as attractive options for new anticancer dr...

The glucose-sensing enzyme glucokinase (GK) plays a key role in glucose metabolism. We report here the effects of a novel glucokinase activator, LY2121260. The activator enhanced GK activity via binding to the allosteric site located in the hinge region of the enzyme. LY2121260 stimulated insulin secretion in a glucose-dependent manner in pancreatic beta-cells and increased glucose use in rat hepatocytes. In addition, incubation of beta-cells with the GK activator resulted in increased GK protein levels, suggesting that enhanced insulin secretion on chronic treatment with a GK activator may be due to not only changed enzyme kinetics but also elevated enzyme levels. Animals treated with LY2121260 showed an improved glucose tolerance after oral glucose challenge. These results support the concept that GK activators represent a new class of compounds that increase both insulin secretion and hepatic glucose use and in doing so may prove to be effective agents for the control of blood glucose levels in patients with type 2 diabetes.

Evidence is presented that the estrogen antagonist 4-hydroxytamoxifen (HT) can occupy not only the core binding pocket within the ligand-binding domain of estrogen receptor (ER) ␤ but also a second site on its surface. The crystal structure of the ligandbinding domain (LBD) associated with HT was determined to 2.2 Å and revealed two molecules of HT bound to the protein. One was located in the consensus ligand-binding pocket, whereas the other bound to a site that overlaps with the hydrophobic groove of the coactivator recognition surface. Relative to the ER␣-tamoxifen structure, helix 12 has been displaced from the coactivator recognition surface and occupies a unique position. Although it has been demonstrated that association of the antagonist with the core ligand-binding pocket is sufficient to induce an antagonist ligandbinding domain conformation, this structure suggests that small molecules may directly antagonize receptor-coactivator interactions. These results provide a direct demonstration of two binding sites for HT in ER␤, as has been previously suggested for ER␣ by using biochemical methods, and represent a crystal structure of a small nonpeptide molecule occupying the coactivator recognition site.antiestrogen ͉ crystallography ͉ nuclear receptor T he estrogen receptor (ER) is a member of the nuclear hormone receptor (NHR) superfamily of ligand-activated transcription factors. Members of this class of proteins display a conserved structural organization consisting of an amino terminal transactivation domain (AF-1), a highly conserved DNAbinding domain, and ligand-binding domain in the carboxyl terminus, which also contains a ligand-activated transactivation function, AF-2 (1). The role that ligands play in modulation of receptor activity has been well characterized by using both biochemical and structural methods and indicates that ligands induce a significant conformational change within the receptor ligand-binding domain (LBD). The structures of several liganded NHRs have been solved, indicating that the ligand binds within the core of the globular LBD. In fact, a ''mouse trap'' model has been proposed (2) in which an agonist accesses the core of the LBD via a pore and, once bound, is ''trapped'' by a conformational shift of a structural component of the LBD itself, helix (H)12. This carboxyl-terminal ␣-helix folds against the surface of the LBD and partially obscures the pore. The conformational shift of H12 also completes the formation of a favorable surface on the LBD that is recognized by transcriptional coactivators that mediate the agonist-dependent transactivation properties of the nuclear receptors. The coactivator recognition surface of the receptor is created by helices 3, 4, 5, and 12 of the LBD and is composed of a hydrophobic groove that is capped on either side by two charged residues (charge clamp), a lysine from H3, and a glutamate from H12 (3, 4). The coactivator motif that is recognized by this groove within the LBD is a conserved amphipathic ␣-helical structure with the consensus...