PDBsum entry 2fsl

Transferase

PDB id: 2fsl

Contents

Protein chain

332 a.a. *

Ligands

BOG

Waters ×207

* Residue conservation analysis

PDB id:

2fsl

Name:

Transferase

Title:

Mitogen activated protein kinase p38alpha (d176a+f327s) activating mutant form-a

Structure:

Mitogen-activated protein kinase 14. Chain: x. Synonym: mitogen-activated protein kinase p38 alpha, map kinase p38 alpha, cytokine suppressive anti-inflammatory drug binding protein, csaid-binding protein, csbp, max-interacting protein 2, map kinase mxi2, sapk2a. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.70Å R-factor: 0.220 R-free: 0.240

Authors:

R.Diskin,O.Livnah

Key ref:

R.Diskin et al. (2007). Structures of p38alpha active mutants reveal conformational changes in L16 loop that induce autophosphorylation and activation. J Mol Biol, 365, 66-76. PubMed id: 17059827 DOI: 10.1016/j.jmb.2006.08.043

Date:

23-Jan-06 Release date: 05-Dec-06

Protein chain

Q16539  (MK14_HUMAN) -  Mitogen-activated protein kinase 14 from Homo sapiens

Seq: 360 a.a.

Struc: 332 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.2.7.11.24 - mitogen-activated protein kinase.
• Reaction:
  1. L-seryl-[protein] + ATP = O-phospho-L-seryl-[protein] + ADP + H⁺
  2. L-threonyl-[protein] + ATP = O-phospho-L-threonyl-[protein] + ADP + H⁺

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1016/j.jmb.2006.08.043

J Mol Biol 365:66-76 (2007)

PubMed id: 17059827

Structures of p38alpha active mutants reveal conformational changes in L16 loop that induce autophosphorylation and activation.

R.Diskin, M.Lebendiker, D.Engelberg, O.Livnah.

ABSTRACT

p38 mitogen-activated protein (MAP) kinases function in numerous signaling processes and are crucial for normal functions of cells and organisms. Abnormal p38 activity is associated with inflammatory diseases and cancers making the understanding of its activation mechanisms highly important. p38s are commonly activated by phosphorylation, catalyzed by MAP kinase kinases (MKKs). Moreover, it was recently revealed that the p38alpha is also activated via alternative pathways, which are MKK independent. The structural basis of p38 activation, especially in the alternative pathways, is mostly unknown. This lack of structural data hinders the study of p38's biology as well as the development of novel strategies for p38 inhibition. We have recently discovered and optimized a novel set of intrinsically active p38 mutants whose activities are independent of any upstream activation. The high-resolution crystal structures of the intrinsically active p38alpha mutants reveal that local alterations in the L16 loop region promote kinase activation. The L16 loop can be thus regarded as a molecular switch that upon conformational changes promotes activation. We suggest that similar conformational changes in L16 loop also occur in natural activation mechanisms of p38alpha in T-cells. Our biochemical studies reveal novel mechanistic insights into the activation process of p38. In this regard, the results indicate that the activation mechanism of the mutants involves dimerization and subsequent trans autophosphorylation on Thr180 (on the phosphorylation lip). Finally, we suggest a model of in vivo p38alpha activation induced by the L16 switch with auto regulatory characteristics.

Selected figure(s)

Figure 1.
Figure 1. Phosphorylation on Thr180 of recombinant p38α^D176A + F327L is temperature-dependent. (a) Kinase assay of p38α^wt and p38α^D176A + F327L purified from cultures grown at different temperatures. The assay measures the ability of the enzymes to phosphorylate in vitro the GST-ATF2 protein substrate using [γ-^32P]ATP. Coomassie staining (upper image) verified the amount of substrate in each lane. The radiograph (lower image) reveals the activity. (b) The active p38α variant is threonine phosphorylated. To elucidate phosphorylation we preformed Western blot analysis utilizing specific anti-phospho antibodies (anti-phospho-Thr, anti-phospho-Tyr and anti-phospho-p38). The small arrows indicate the migration height of p38α in gel electrophoresis. Anti-p38α antibody was used to determine the amount of p38 loaded at each lane. Some minor phosphorylation on tyrosine residues can be seen only on smaller proteolytic products (marked with asterisks). Yet, these proteins also interacted with the anti-phospho-p38 antibody. (c) The p38α^D176A + F327L molecule exhibits autophosphorylation in vitro. Purified p38α^D176A + F327L that was also used for crystallization was incubated in a kinase assay buffer without substrate for increasing time intervals at 30 °C. Coomassie staining (upper image) verified the amount of enzyme in each lane. The radiograph (lower image) reveals phosphorylation. (d) Mono-phosphorylated p38α is catalytically active. We measured kinase activity toward GST-ATF2 of p38α^Y182F, p38α^T180A and p38α^wt activated or not in vitro by MKK6. Coomassie staining (upper image) verified the amount of GST-ATF2 in each lane. The radiograph (lower image) indicates activity. Figure 1. Phosphorylation on Thr180 of recombinant p38α^D176A + F327L is temperature-dependent. (a) Kinase assay of p38α^wt and p38α^D176A + F327L purified from cultures grown at different temperatures. The assay measures the ability of the enzymes to phosphorylate in vitro the GST-ATF2 protein substrate using [γ-^32P]ATP. Coomassie staining (upper image) verified the amount of substrate in each lane. The radiograph (lower image) reveals the activity. (b) The active p38α variant is threonine phosphorylated. To elucidate phosphorylation we preformed Western blot analysis utilizing specific anti-phospho antibodies (anti-phospho-Thr, anti-phospho-Tyr and anti-phospho-p38). The small arrows indicate the migration height of p38α in gel electrophoresis. Anti-p38α antibody was used to determine the amount of p38 loaded at each lane. Some minor phosphorylation on tyrosine residues can be seen only on smaller proteolytic products (marked with asterisks). Yet, these proteins also interacted with the anti-phospho-p38 antibody. (c) The p38α^D176A + F327L molecule exhibits autophosphorylation in vitro. Purified p38α^D176A + F327L that was also used for crystallization was incubated in a kinase assay buffer without substrate for increasing time intervals at 30 °C. Coomassie staining (upper image) verified the amount of enzyme in each lane. The radiograph (lower image) reveals phosphorylation. (d) Mono-phosphorylated p38α is catalytically active. We measured kinase activity toward GST-ATF2 of p38α^Y182F, p38α^T180A and p38α^wt activated or not in vitro by MKK6. Coomassie staining (upper image) verified the amount of GST-ATF2 in each lane. The radiograph (lower image) indicates activity.

Figure 3.
Figure 3. Conformational changes in the L16 loop and disruption of a salt-bridge are fingerprints of the active p38α molecules. (a) The conformational changes within the L16 loops of p38α^D176A + F327L (blue) and p38α^D176A + F327S (yellow) in reference to p38α^D176A (magenta) and p38α^wt (gray). Mutations of Phe327 to serine or leucine results in a conformational change in the L16 loop. Residues 327 in the mutants' structures subsequently adopt a different conformation. However, the conformation of Trp337 and Tyr69 remains highly similar in all structures. (b) Segments of L16 loop from p38α^D176A (left), p38α^D176A + F327L (center) and p38α^D176A + F327S (right) are superimposed with p38α^wt as a reference. The conformation of the L16 loop in the structure of p38α^D176A is almost identical (except minor changes in Asp331) to that of p38α^wt. Mutation of Phe327 leads to the unwinding and a shift of the main-chain helical conformation in the L16, and subsequently the side-chains of residues 324 to 330 adopt a different position in both p38α^D176A + F327L and p38α^D176A + F327S models (center and right). (c) A salt bridge interaction is formed between the negatively charged carboxyl group of Glu328 and the positively charged guanidine of Arg70 (green broken lines) in both p38α^wt and p38α^D176A (left). This salt bridge is disrupted in the structures of p38α^D176A + F327L and p38α^D176A + F327S (center and right, respectively) due to the conformational change in the L16 loop. In this regard, the unpaired Arg70 acquire new conformations; in p38α^D176A + F327L Arg70 adopts a dual conformation whereas in p38α^D176A + F327S only one. The C^α atom of Glu328 is shifted 2.53 Å and 1.11 Å in the structures of p38α^D176A + F327L and p38α^D176A + F327S, respectively, relative to the p38α^wt structure. The orientation of the side-chains is somewhat different as Lys66 is stabilizing the carbonyl oxygen of Glu328 by forming an H-bond interaction in p38α^D176A + F327S similar to p38α^wt but not in p38α^D176A + F327L (yellow broken lines). Figure 3. Conformational changes in the L16 loop and disruption of a salt-bridge are fingerprints of the active p38α molecules. (a) The conformational changes within the L16 loops of p38α^D176A + F327L (blue) and p38α^D176A + F327S (yellow) in reference to p38α^D176A (magenta) and p38α^wt (gray). Mutations of Phe327 to serine or leucine results in a conformational change in the L16 loop. Residues 327 in the mutants' structures subsequently adopt a different conformation. However, the conformation of Trp337 and Tyr69 remains highly similar in all structures. (b) Segments of L16 loop from p38α^D176A (left), p38α^D176A + F327L (center) and p38α^D176A + F327S (right) are superimposed with p38α^wt as a reference. The conformation of the L16 loop in the structure of p38α^D176A is almost identical (except minor changes in Asp331) to that of p38α^wt. Mutation of Phe327 leads to the unwinding and a shift of the main-chain helical conformation in the L16, and subsequently the side-chains of residues 324 to 330 adopt a different position in both p38α^D176A + F327L and p38α^D176A + F327S models (center and right). (c) A salt bridge interaction is formed between the negatively charged carboxyl group of Glu328 and the positively charged guanidine of Arg70 (green broken lines) in both p38α^wt and p38α^D176A (left). This salt bridge is disrupted in the structures of p38α^D176A + F327L and p38α^D176A + F327S (center and right, respectively) due to the conformational change in the L16 loop. In this regard, the unpaired Arg70 acquire new conformations; in p38α^D176A + F327L Arg70 adopts a dual conformation whereas in p38α^D176A + F327S only one. The C^α atom of Glu328 is shifted 2.53 Å and 1.11 Å in the structures of p38α^D176A + F327L and p38α^D176A + F327S, respectively, relative to the p38α^wt structure. The orientation of the side-chains is somewhat different as Lys66 is stabilizing the carbonyl oxygen of Glu328 by forming an H-bond interaction in p38α^D176A + F327S similar to p38α^wt but not in p38α^D176A + F327L (yellow broken lines).

The above figures are reprinted by permission from Elsevier: J Mol Biol (2007, 365, 66-76) copyright 2007.

Figures were selected by an automated process.