X-ray structure of the human mitogen-activated protein kinase kinase 2 (MEK2)in a complex with ligand and MgATP (original) (raw)
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High-resolution crystal structure of human Mapkap kinase 3 in complex with a high affinity ligand
Protein Science, 2009
The Mapkap kinases 2 and 3 (MK2 and MK3) have been implicated in intracellular signaling pathways leading to the production of the pro-inflammatory cytokine tumor necrosis factor alpha. MK2 has been pursued by the biopharmaceutical industry for many years for the development of a small molecule anti-inflammatory treatment and drug-like inhibitors have been described. The development of some of these compounds, however, has been slowed by the absence of a high-resolution crystal structure of MK2. Herein we present a high-resolution (1.9 Å) crystal structure of the highly homologous MK3 in complex with a pharmaceutical lead compound. While all of the canonical features of Ser/Thr kinases in general and MK2 in particular are recapitulated in MK3, the detailed analysis of the binding interaction of the drug-like ligand within the adenine binding pocket allows relevant conclusions to be drawn for the further design of potent and selective drug candidates.
Biochemical Journal, 1993
The substrate specificity of mitogen-activated protein (MAP) kinase-activated protein kinase-2 (MAPKAP kinase-2) was investigated by using synthetic peptides related to the N-terminus of glycogen synthase. The minimum sequence required for efficient phosphorylation was found to be Xaa-Xaa-Hyd-Xaa-Arg-Xaa-Xaa-Ser-Xaa-Xaa, where Hyd is a bulky hydrophobic residue (Phe > Leu > Val >> Ala), and the peptide Lys-Lys-Phe-Asn-Arg-Thr-Leu-Ser-Val-Ala was phosphorylated with a Km of 9.3 microM and Vmax. of 10 mumol/min per mg. MAPKAP kinase-1 (a homologue of ribosomal protein S6 kinase) also requires an arginine three residues N-terminal to the serine (position n-3), but not a hydrophobic residue at position n-5. Neither MAPKAP kinase-1 nor MAPKAP kinase-2 could tolerate a proline residue at position n + 1, indicating that their specificities do not overlap with that of MAP kinase. The specificity of calmodulin-dependent protein kinase-II resembled that of MAPKAP kinase-2, except ...
Structure, 2005
Here, we present the 2.1 Å crystal structure of a nonphosphorylated Mnk2 fragment that encompasses the kinase domain. The results show Mnk-specific features such as a zinc binding motif and an atypical open conformation of the activation segment. In addition, the ATP binding pocket contains an Asp-Phe-Asp (DFD) in place of the canonical magnesium binding Asp-Phe-Gly (DFG) motif. The phenylalanine of this motif sticks into the ATP binding pocket and blocks ATP binding as observed with inhibitor bound and, thus, inactive p38 kinase. Replacement of the DFD by the canonical DFG motif affects the conformation of Mnk2, but not ATP binding and kinase activity. The results suggest that the ATP binding pocket and the activation segment of Mnk2 require conformational switches to provide kinase activity. References Adams, J.A. (2001). Kinetic and catalytic mechanisms of protein kinases. Chem. Rev. 101, 2271-2290. Arquier, N., Bourouis, M., Colombani, J., and Leopold, P. (2005). Drosophila Lk6 kinase controls phosphorylation of eukaryotic translation initiation factor 4E and promotes normal growth and development. Curr. Biol. 15, 19-23. Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., et al. (1998). Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905-921. CCP4 (Collaborative Computational Project, Number 4) (1994). The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760-763. Ueda, T., Watanabe-Fukunaga, R., Fukuyama, H., Nagata, S., and Fukunaga, R. (2004). Mnk2 and Mnk1 are essential for constitutive and inducible phosphorylation of eukaryotic initiation factor 4E but not for cell growth or development. Mol. Cell. Biol. 24, 6539-6549. von der Haar, T., Gross, J.D., Wagner, G., and McCarthy, J.E. (2004). The mRNA cap-binding protein eIF4E in post-transcriptional gene expression. Nat. Struct. Mol. Biol. 11, 503-511. Waskiewicz, A.J., Flynn, A., Proud, C.G., and Cooper, J.A. (1997). Mitogen-activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2. EMBO J. 16, 1909-1920. Waskiewicz, A.J., Johnson, J.C., Penn, B., Mahalingam, M., Kimball, S.R., and Cooper, J.A. (1999). Phosphorylation of the capbinding protein eukaryotic translation initiation factor 4E by protein kinase Mnk1 in vivo. . Crystal structure of p38 mitogen-activated protein kinase. J. Biol. Chem. 271, 27696-27700.
The Structure of the MAP2K MEK6 Reveals an Autoinhibitory Dimer
Structure, 2009
The mitogen-activated protein (MAP) kinase protein family has a critical role in cellular signaling events, with MAP kinase p38α 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 p38α 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)-1Hpyrazol-4-yl]pyridine, to wild-type p38α induces movement of the Cterminal 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 p38α 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 p38α in predetermined ways. Moreover, these results and analyses of p38α suggest strategies for designing specific binding compounds applicable to other MAP kinases, as well as the cyclin-dependent kinase family and glycogen synthase kinase 3β that also utilize the C-terminal insert in their interactions.
X-ray structure of a bifunctional protein kinase in complex with its protein substrate HPr
Proceedings of The National Academy of Sciences, 2002
X-Ray Diffraction Data Collection. Data collection was performed at 100 K on flash-frozen crystals at European Synchrotron Radiation Facility (Grenoble, France) on stations ID14-H2 for the HPr complex and BM14 for the P-Ser-HPr complex. Images were processed with DENZO, and data were scaled with SCALE-PACK (17). Statistics are given in . The crystals of the two complexes are isomorphous and belong to space group P3 2 12 This paper was submitted directly (Track II) to the PNAS office.
The Crystal Structure of Cancer Osaka Thyroid Kinase Reveals an Unexpected Kinase Domain Fold
Journal of Biological Chemistry, 2015
Background: Cancer Osaka Thyroid (COT) kinase plays a crucial role in inflammatory diseases and cancer. Results: Production of catalytically competent COT kinase yielded protein suitable for structure guided drug discovery. Conclusion: COT kinase has a unique and structurally versatile active site. Significance: The discovery of a novel variation of the protein kinase fold will impact drug discovery for COT kinase.
The EMBO Journal, 2006
Autoinhibition is a recurring mode of protein kinase regulation and can be based on diverse molecular mechanisms. Here, we show by crystal structure analysis, nuclear magnetic resonance (NMR)-based nucleotide affinity studies and rational mutagenesis that nonphosphorylated mitogen-activated protein (MAP) kinases interacting kinase (Mnk) 1 is autoinhibited by conversion of the activation segment into an autoinhibitory module. In a Mnk1 crystal structure, the activation segment is repositioned via a Mnk-specific sequence insertion at the N-terminal lobe with the following consequences: (i) the peptide substrate binding site is deconstructed, (ii) the interlobal cleft is narrowed, (iii) an essential Lys-Glu pair is disrupted and (iv) the magnesium-binding loop is locked into an ATP-competitive conformation. Consistently, deletion of the Mnk-specific insertion or removal of a conserved phenylalanine side chain, which induces a blockade of the ATP pocket, increase the ATP affinity of Mnk1. Structural rearrangements required for the activation of Mnks are apparent from the cocrystal structure of a Mnk2 D228Gstaurosporine complex and can be modeled on the basis of crystal packing interactions. Our data suggest a novel regulatory mechanism specific for the Mnk subfamily.
Structural Basis for a High Affinity Inhibitor Bound to Protein Kinase MK2
Journal of Molecular Biology, 2007
The Ser/Thr protein kinase MAPKAP kinase 2 (MK2) plays a crucial role in inflammation. We determined the structure of the kinase domain of MK2 in complex with a low molecular mass inhibitor in two different crystal forms, obtained from soaking and co-crystallization. To our knowledge, these are the first structures of MK2 showing the binding mode of an inhibitor with high binding affinity (IC50 8.5 nM). The two crystal forms revealed conformational flexibility in the binding site and extend the experimental basis for rational drug design. Crystal form-1 contained one MK2 molecule per asymmetric unit. Form-2 contained 12 molecules, which arrange into two different types of MK2 trimers. One of them may serve as a model for an intermediate state during substrate phosphorylation, as each MK2 monomer places its activation segment into the substrate peptide binding groove of the trimer neighbor.
Bioorganic & Medicinal Chemistry Letters, 2009
An approach and preliminary results for utilizing legacy MEK inhibitors as templates for a reiterative structural based design and synthesis of novel, type III NCKIs (non-classical kinase inhibitors) is described. Evidence is provided that the MEK-pocket or pockets closely related to it may exist in kinases other than MEK. and related analogs are potent and exquisitely selective MEK kinase inhibitors. CI-1040 and PD-0325901 have advanced to the clinic. 1-5 The inhibitors are non-ATP competitive and bind to a unique pocket (designated hence forth as the M-pocket) adjacent to, but distinct from, the ATP binding pocket.