Dataset Information

P38alpha MAP kinase C-terminal domain binding pocket characterized by crystallographic and computational analyses.

ABSTRACT: The mitogen-activated protein (MAP) kinase protein family has a critical role in cellular signaling events, with MAP kinase p38alpha acting in inflammatory processes and being an important drug discovery target. MAP kinase drug design efforts have focused on small-molecule inhibitors of the ATP catalytic site, which exhibit dose-limiting adverse effects. Therefore, characterizing other potential sites that bind substrates, inhibitors, or allosteric effectors is of great interest. Here, we present the crystal structure of human p38alpha MAP kinase, which has a lead compound bound both in the active site and in the lipid-binding site of the C-terminal cap. This C-terminal cap is formed from an extension to the kinase fold, unique to the MAP kinase and cyclin-dependent kinase families and glycogen synthase kinase 3. Binding of this lead, 4-[3-(4-fluorophenyl)-1H-pyrazol-4-yl]pyridine, to wild-type p38alpha induces movement of the C-terminal cap region, creating a hydrophobic pocket centered around residue Trp197. Computational analysis of this C-terminal domain pocket indicates notable flexibility for potentially binding different-shaped compounds, including lipids, oxidized arachidonic acid species such as leukotrienes, and small-molecule effectors. Furthermore, our structural results defining the open p38alpha C-lobe pocket provide a detailed framework for the design of novel small molecules with affinities comparable to active-site binders: to bind and potentially modulate the shape and activity of p38alpha in predetermined ways. Moreover, these results and analyses of p38alpha suggest strategies for designing specific binding compounds applicable to other MAP kinases, as well as the cyclin-dependent kinase family and glycogen synthase kinase 3beta that also utilize the C-terminal insert in their interactions.

SUBMITTER: Perry JJ

PROVIDER: S-EPMC2724755 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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p38alpha MAP kinase C-terminal domain binding pocket characterized by crystallographic and computational analyses.

Perry J Jefferson P JJ Harris Rodney M RM Moiani Davide D Olson Arthur J AJ Tainer John A JA

Journal of molecular biology 20090606 1

The mitogen-activated protein (MAP) kinase protein family has a critical role in cellular signaling events, with MAP kinase p38alpha acting in inflammatory processes and being an important drug discovery target. MAP kinase drug design efforts have focused on small-molecule inhibitors of the ATP catalytic site, which exhibit dose-limiting adverse effects. Therefore, characterizing other potential sites that bind substrates, inhibitors, or allosteric effectors is of great interest. Here, we presen ...[more]

PMID: 19501598

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Project description:BACKGROUND: Human epidermal growth factor receptor 2 (HER2) has an important role in cancer aggressiveness and poor prognosis. HER2 has been used as a drug target for cancers. In particular, to effectively treat HER2-positive cancer, small molecule inhibitors were developed to target HER2 kinase. Knowing that curcumin has been used as food to inhibit cancer activity, this study evaluated the efficacy of natural curcumins and curcumin analogs as HER2 inhibitors using in vitro and in silico studies. The curcumin analogs considered in this study composed of 4 groups classified by their core structure, ?-diketone, monoketone, pyrazole, and isoxazole. RESULTS: In the present study, both computational and experimental studies were performed. The specificity of curcumin analogs selected from the docked results was examined against human breast cancer cell lines. The screened curcumin compounds were then subjected to molecular dynamics simulation study. By modifying curcumin analogs, we found that protein-ligand affinity increases. The benzene ring with a hydroxyl group could enhance affinity by forming hydrophobic interactions and the hydrogen bond with the hydrophobic pocket. Hydroxyl, carbonyl or methoxy group also formed hydrogen bonds with residues in the adenine pocket and sugar pocket of HER2-TK. These modifications could suggest the new drug design for potentially effective HER2-TK inhibitors. Two outstanding compounds, bisdemethylcurcumin (AS-KTC006) and 3,5-bis((E)-3,4-dimethoxystyryl)isoxazole (AS-KTC021 ),were well oriented in the binding pocket almost in the simulation time, 30 ns. This evidence confirmed the results of cell-based assays and the docking studies. They possessed more distinguished interactions than known HER2-TK inhibitors, considering them as a promising drug in the near future. CONCLUSIONS: The series of curcumin compounds were screened using a computational molecular docking and followed by human breast cancer cell lines assay. Both AS-KTC006 and AS-KTC021 could inhibit breast cancer cell lines though inhibiting of HER2-TK. The intermolecular interactions were confirmed by molecular dynamics simulation studies. This information would explore more understanding of curcuminoid structures and HER2-TK.

Project description:Hydrogen-deuterium exchange measurements represent a powerful approach to investigating changes in conformation and conformational mobility in proteins. Here, we examine p38alpha MAP kinase (MAPK) by hydrogen-exchange (HX) mass spectrometry to determine whether changes in conformational mobility may be induced by kinase phosphorylation and activation. Factors influencing sequence coverage in the HX mass spectrometry experiment, which show that varying sampling depths, instruments, and peptide search strategies yield the highest coverage of exchangeable amides, are examined. Patterns of regional deuteration in p38alpha are consistent with tertiary structure and similar to deuteration patterns previously determined for extracellular-signal-regulated kinase (ERK) 2, indicating that MAPKs are conserved with respect to the extent of local amide HX. Activation of p38alpha alters HX in five regions, which are interpreted by comparing X-ray structures of unphosphorylated p38alpha and X-ray structures of phosphorylated p38gamma. Conformational differences account for altered HX within the activation lip, the P+1 site, and the active site. In contrast, HX alterations are ascribed to activation-induced effects on conformational mobility, within substrate-docking sites (alphaF-alphaG, beta7-beta8), the C-terminal core (alphaE), and the N-terminal core region (beta4-beta5, alphaL16, alphaC). Activation also decreases HX in a 3-10 helix at the C-terminal extension of p38alpha. Although this helix in ERK2 forms a dimerization interface that becomes protected from HX upon activation, analytical ultracentrifugation shows that this does not occur in p38alpha because both unphosphorylated and diphosphorylated forms are monomeric. Finally, HX patterns in monophosphorylated p38alpha are similar to those in unphosphorylated kinase, indicating that the major activation lip remodeling events occur only after diphosphorylation. Importantly, patterns of activation-induced HX show differences between p38alpha and ERK2 despite their similarities in overall deuteration, suggesting that although MAPKs are closely related with respect to primary sequence and tertiary structure, they have distinct mechanisms for dynamic control of enzyme function.

Dataset Information

P38alpha MAP kinase C-terminal domain binding pocket characterized by crystallographic and computational analyses.

Publications

p38alpha MAP kinase C-terminal domain binding pocket characterized by crystallographic and computational analyses.

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