The Structure of the MAP2K MEK6 Reveals an Autoinhibitory Dimer (original) (raw)
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Journal of Molecular Biology, 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.
Journal of Medicinal Chemistry, 2007
The main recognition characteristics of the ATP binding site of p38 mitogen activated protein kinase alpha (p38R MAPK) have been explored by a combination of modeling and bioinformatics techniques, making special emphasis in the characteristics of the site that justifies binding specificity with respect to other MAP kinases. Particularly, we have analyzed the binding mode of a new family of p38 MAPK inhibitors based on the pyridinyl-heterocycle core. This family of compounds has a marked pseudosymmetry and can exist in different tautomeric forms, which makes the determination of the binding mode especially challenging. A combination of homology modeling, quantum mechanics, classical docking, and molecular dynamics calculations allowed us to determine the main characteristics defining the binding mode of this new scaffold in the ATP binding site of p38R. A set of free energy calculations allowed us to verify the binding mode proposed, giving an overall excellent agreement with the experimental values. Finally, the binding mode of this new family of compounds was compared to that of other members of the pyridinyl and pyrimidinyl heterocycle class. (results of quantum mechanical calculations on the different tautomeric species) and graph S1 (theoretical vs experimental relative free energies of binding for compound 13 and derivatives). This material is available free of charge via the Internet at http://pubs.acs.org.
Molecular cell, 2002
The structures of the MAP kinase p38 in complex with docking site peptides containing a phi(A)-X-phi(B) motif, derived from substrate MEF2A and activating enzyme MKK3b, have been solved. The peptides bind to the same site in the C-terminal domain of the kinase, which is both outside the active site and distinct from the "CD" domain previously implicated in docking site interactions. Mutational analysis on the interaction of p38 with the docking sites supports the crystallographic models and has uncovered two novel residues on the docking groove that are critical for binding. The two peptides induce similar large conformational changes local to the peptide binding groove. The peptides also induce unexpected and different conformational changes in the active site, as well as structural disorder in the phosphorylation lip.
Acta crystallographica. Section D, Biological crystallography, 2015
The p38 MAP kinases (p38 MAPKs) represent an important family centrally involved in mediating extracellular signaling. Recent studies indicate that family members such as MAPK13 (p38δ) display a selective cellular and tissue expression and are therefore involved in specific diseases. Detailed structural studies of all p38 MAPK family members are crucial for the design of specific inhibitors. In order to facilitate such ventures, the structure of MAPK13 was determined in both the inactive (unphosphorylated; MAPK13) and active (dual phosphorylated; MAPK13/pTpY) forms. Here, the first preparation, crystallization and structure determination of MAPK13/pTpY are presented and the structure is compared with the previously reported structure of MAPK13 in order to facilitate studies for structure-based drug design. A comprehensive analysis of inactive versus active structures for the p38 MAPK family is also presented. It is found that MAPK13 undergoes a larger interlobe configurational rearr...
A Novel Lipid Binding Site Formed by the MAP Kinase Insert in p38α
Journal of Molecular Biology, 2008
The p38 mitogen-activated protein (MAP) kinases function as signaling molecules essential for many cellular processes, particularly mediating stress response. The activity of p38 MAP kinases is meticulously regulated to reach the desired cellular phenotype. Several alternative activation and attenuation mechanisms have been characterized recently which include new phosphorylation sites. Here we present the crystal structure of p38α MAP kinase in complex with n-octyl-β-glucopyranoside detergent. The complex unveils a novel lipid-binding site formed by a local conformational change of the MAP kinase insert. This binding is the first attribution for a possible role of the MAP kinase insert in p38. The binding site can accommodate a large selection of lipidic molecules. In addition, we also show via biophysical methods that arachidonic acid and its derivatives bind p38α in vitro. Based on our analysis we propose that the binding of lipids could fine-tune p38α catalytic activity towards a preferred phenotype.
Effect of the distal C162S mutation on the energetics of drug binding to p38α MAP kinase
Archives of Biochemistry and Biophysics, 2008
The binding reactions of the inhibitor drugs, SB 203580, SKF 86002, and p38 INH.1 to the isoforms 1 and 2 splice variants of p38a MAP kinase and their C162S mutants, as determined from ITC measurements from 25 to 35°C, are totally enthalpically driven with binding constants ranging from 10 7 M À1 for SKF 86002 and SB 203580 to 10 9 M À1 for p38 INH.1. Interactions of p38 INH.1 with an additional hydrophobic pocket of the kinase would account for its large increase in K b . DSC scans exhibited single unfolding transitions for the isoforms, their mutants, and the mutants bound to the drug inhibitors. Two transitions, however, were observed for the isoform-drug complexes of SB 203580 and p38 INH.1 and were attributed to decoupled unfolding of the N-and C-terminal domains of the kinase. The C-terminal domain of isoform 1 is estimated to be less stable than of isoform 2 by 15 kJ mol À1 . piperazine-N 0 (2-ethanesulfonic acid); isoform 1, p38a MAP kinase isoform 1 splice variant; isoform 1 C162S, p38a MAP kinase isoform 1 with C162S mutation; isoform 2, wild type p38a MAP kinase; isoform 2 C162S, p38a MAP kinase isoform 2 splice variant with C162S mutation; ITC, isothermal titration calorimetry; p38 INH.1, 1-(2,6-dichlorophenyl)-5-(2,4-difluorophenyl)-7-piperidine-4-yl-3,4-dihydroquinoline-2(1H)-one; SB 203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl) 1H-imidazole; SKF 86002, 6-(4-fluorophenyl)-2 , 3 -d i h y d r o -5 -( 4 -p y r i d y l ) i m i d a z o -[ 2 , 1b ] -t h i a z o l e ; T r i s , tris[hydroxymethyl]aminomethane.
Structural basis for p38α MAP kinase quinazolinone and pyridol-pyrimidine inhibitor specificity
Nature Structural Biology, 2003
Mitogen-activated protein kinases (MAPKs) are constituents of numerous signal transduction pathways that control complex processes such as differentiation, proliferation and cell death. They are also involved in signaling pathways that respond rapidly to environmental changes, such as those required for homeostasis and acute hormonal responses 1-4 . Members of the MAP kinase family share sequence similarity and conserved structural motifs, and are all activated by dual phosphorylation of conserved threonine and tyrosine residues in the activation loop. However, the various MAP kinases are responsive to different extracellular stimuli, and each activates a unique, although overlapping, spectrum of cellular targets. Whereas the extracellular signal-regulated kinases (ERKs) are activated in response to hormones and growth factors, the c-Jun N-terminal kinases (JNKs) and p38α are activated by lipopolysaccharide (LPS) and by environmental stresses such as heat shock, hyperosmolarity and radiation 5-9 .
Acta Crystallographica Section D Biological Crystallography, 2009
PDB References: MAP kinase p38, inhibitor complex, 3gc7, r3gc7sf; MAP kinase p38, C162S mutant, 3gc8, r3gc8sf; C119S,C162S mutant, 3gc9, r3gc9sf.
Structural basis for p38alpha MAP kinase quinazolinone and pyridol-pyrimidine inhibitor specificity
Nature structural biology, 2003
The quinazolinone and pyridol-pyrimidine classes of p38 MAP kinase inhibitors have a previously unseen degree of specificity for p38 over other MAP kinases. Comparison of the crystal structures of p38 bound to four different compounds shows that binding of the more specific molecules is characterized by a peptide flip between Met109 and Gly110. Gly110 is a residue specific to the alpha, beta and gamma isoforms of p38. The delta isoform and the other MAP kinases have bulkier residues in this position. These residues would likely make the peptide flip energetically unfavorable, thus explaining the selectivity of binding. To test this hypothesis, we constructed G110A and G110D mutants of p38 and measured the potency of several compounds against them. The results confirm that the selectivity of quinazolinones and pyridol-pyrimidines results from the presence of a glycine in position 110. This unique mode of binding may be exploited in the design of new p38 inhibitors.