The structural basis for the divalent cation-dependent binding of heterodimeric alphabeta integrins to their ligands, which contain the prototypical Arg-Gly-Asp sequence, is unknown. Interaction with ligands triggers tertiary and quaternary structural rearrangements in integrins that are needed for cell signaling. Here we report the crystal structure of the extracellular segment of integrin alphaVbeta3 in complex with a cyclic peptide presenting the Arg-Gly-Asp sequence. The ligand binds at the major interface between the alphaV and beta3 subunits and makes extensive contacts with both. Both tertiary and quaternary changes are observed in the presence of ligand. The tertiary rearrangements take place in betaA, the ligand-binding domain of beta3; in the complex, betaA acquires two cations, one of which contacts the ligand Asp directly and the other stabilizes the ligand-binding surface. Ligand binding induces small changes in the orientation of alphaV relative to beta3.

We have investigated the role of subcellular localization in the regulation of protein kinase B (PKB) activation. The myristoylation/palmitylation motif from the Lck tyrosine kinase was attached to the N terminus of protein kinase B to alter its subcellular location. Myristoylated/palmitylated (m/p)-PKB␣ was associated with the plasma membrane of transfected cells, whereas the wild-type kinase was mostly cytosolic. The activity of m/p-PKB␣ was 60-fold higher compared with the unstimulated wild-type enzyme, and could not be stimulated further by growth factors or phosphatase inhibitors. In vivo 32 P labeling and mutagenesis demonstrated that m/p-PKB␣ activity was due to phosphorylation on ). Three mammalian isoforms of PKB have been identified so far, termed PKB␣, -␤, and -␥ (7-9). 2 All three isoforms contain a pleckstrin homology (PH) domain at the N terminus (10), followed by a catalytic domain related to protein kinases A and C, and a C-terminal regulatory region. PKB␣ was found to mediate insulin-and insulin-like growth factor (IGF-1)-induced cellular responses, such as the inhibition of glycogen synthase kinase-3 (11), the stimulation of glucose uptake (12), and the promotion of cell survival by inhibiting apoptosis (Ref. 13; reviewed in Refs. 14 and 15). PKB␣ is the cellular homologue of the oncogene product v-Akt encoded by the AKT8 retrovirus, which induces thymic lymphomas in mice (16). Cloning of v-akt revealed that it was created by fusion of viral Gag sequences to the N terminus of mouse PKB␣, which adds an N-terminal myristoylation signal to the oncoprotein and could account for its transforming ability (2, 17). Overexpression of PKB␣ or -␤ is associated with some human ovarian, pancreatic, and breast carcinomas (8, 18 -20).PKB␣ is activated by a variety of growth factors and phosphatase inhibitors (5, 6, 21) through a phosphorylation mechanism (21-23). The activation of PKB␣ by insulin or IGF-1 is mediated by phosphorylation of Thr 308 in the catalytic domain and Ser 473 at the C terminus (22). The phosphorylation of both sites is blocked by pretreatment of the cells with the PI3-K inhibitor wortmannin. Substitution of both regulatory sites by aspartic acid residues to mimic phosphorylation by the introduction of a negative charge, produces a constitutively active enzyme (22). This work predicted the existence of an upstream kinase(s) that phosphorylate(s) these sites, and recently a protein kinase activity was identified and purified capable of phosphorylating Thr 308 in the presence of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) or phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P 2 ) (Refs. 24 and 25; reviewed in Ref. 26). The enzyme has therefore been termed 3-phosphoinositide-dependent protein kinase-1 (PDK1).The PH domain of PKB has been reported to play a role in the activation process (6), but PKB activation can also occur in its absence, depending on the agonist and the type of deletion mutants used (21,23,27). The PH domain of PKB binds PtdIns(3,4,5)P 3 and PtdIns(3,4)P...

Drug-target residence time (τ), one of the main determinants of drug efficacy, remains highly challenging to predict computationally and, therefore, is usually not considered in the early stages of drug design. Here, we present an efficient computational method, τ-random acceleration molecular dynamics (τRAMD), for the ranking of drug candidates by their residence time and obtaining insights into ligand-target dissociation mechanisms. We assessed τRAMD on a data set of 70 diverse drug-like ligands of the N-terminal domain of HSP90α, a pharmaceutically important target with a highly flexible binding site, obtaining computed relative residence times with an accuracy of about 2.3τ for 78% of the compounds and less than 2.0τ within congeneric series. Analysis of dissociation trajectories reveals features that affect ligand unbinding rates, including transient polar interactions and steric hindrance. These results suggest that τRAMD will be widely applicable as a computationally efficient aid to improving drug residence times during lead optimization.

The influence of inositol phosphates and phosphoinositides on the ␣ isoform of the RAC-protein kinase B (RAC/PKB) was studied using purified wild type and mutant kinase preparations and a recombinant pleckstrin homology (PH) domain. Binding of inositol phosphates and phosphoinositides to the PH domain was measured as the quenching of intrinsic tryptophan fluorescence. Inositol phosphates and D3-phosphorylated phosphoinositides bound with affinities of 1-10 M and 0.5 M, respectively. Similar values were obtained using RAC/PKB expressed and purified from baculovirus-infected Sf9 cells in the fluorescence assay. The influence of synthetic dioctanoyl derivatives of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate on the activity of RAC/PKB purified from transfected COS-1 cells was studied. Phosphatidylinositol 3,4,5-trisphosphate was found to inhibit the RAC/ PKB kinase activity with half-maximal inhibition at 2.5 M. In contrast, phosphatidylinositol 3,4-bisphosphate stimulated kinase activity (half-maximal stimulation at 2.5 M). A mutant RAC/PKB protein lacking the PH domain was not affected by D3-phosphorylated phosphoinositides. These results demonstrate that the PH domain of RAC/PKB binds inositol phosphates and phosphoinositides with high affinity, and suggest that the products of the phosphatidylinositide 3-kinase can act as both a membrane anchor and modulator of RAC/ PKB activity. The data also provide further evidence for a link between phosphatidylinositide 3-kinase and RAC/ PKB regulation. The stimulation of receptor tyrosine kinases (RTK)1 by agonists leads to immediate activation of intracellular signal transduction pathways. The assembly of multiprotein complexes at the plasma membrane is one important feature of RTK signal transduction mechanisms (reviewed in Refs. 1 and 2). Numerous studies suggest that the activation of phosphatidylinositide 3-kinase (PI3-K) by growth factors is also involved (3, 4), leading to the accumulation of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P 2 ); these metabolites are assumed to act as second messengers (5). Recently RAC-protein kinase B (RAC/PKB) has emerged as a key player in the PI3-K-stimulated signaling pathway, based on the inhibition of its activation by wortmannin (6 -11).RAC/PKB is a subfamily of the second messenger-regulated serine/threonine kinases (12). Three isoforms (␣, ␤, ␥) have been identified, each consisting of an amino-terminal pleckstrin homology (PH) domain, a central kinase domain, and a serine/threonine-rich carboxyl-terminal region (13-17). Various stimuli, such as insulin, PDGF, epidermal growth factor, basic fibroblast growth factor, serum (6 -10), and protein phosphatase inhibitors (9), lead to activation of RAC/PKB kinase activity. The activation is promoted by signals emanating from RTK-regulated PI3-K and is accompanied by an increase in serine/threonine phosphorylation of RAC/PKB (6, 9). The phosphorylation of two sites has been ...

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