Mutagenesis of p38α MAP Kinase Establishes Key Roles of Phe169 in Function and Structural Dynamics and Reveals a Novel DFG-OUT State

Bukhtiyarova, Marina; Karpusas, Michael; Northrop, Katrina; Namboodiri, H.V.; Springman, Eric B.

doi:10.1021/bi0622221

Cited by 34 publications

(38 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our simulations also show that the DFG flip involves extensive backbone-torsion motions at the glycine residue, suggesting that the unique flexibility of glycine may serve to lower the kinetic barrier for the flip. This is consistent with work (41) suggesting that the DFG motif is optimized for functional dynamics rather than for structural stability, as is our finding that the DFG-out conformation can be populated under physiological conditions even in the absence of DFG-out binders.…”

Section: Conservation Of the Dfg Motif And Its Role In Kinase Catalysissupporting

confidence: 92%

A conserved protonation-dependent switch controls drug binding in the Abl kinase

Shan

Seeliger

Eastwood

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

239

314

View full text Add to dashboard Cite

In many protein kinases, a characteristic conformational change (the ''DFG flip'') connects catalytically active and inactive conformations. Many kinase inhibitors-including the cancer drug imatinib-selectively target a specific DFG conformation, but the function and mechanism of the flip remain unclear. Using long molecular dynamics simulations of the Abl kinase, we visualized the DFG flip in atomiclevel detail and formulated an energetic model predicting that protonation of the DFG aspartate controls the flip. Consistent with our model's predictions, we demonstrated experimentally that the kinetics of imatinib binding to Abl kinase have a pH dependence that disappears when the DFG aspartate is mutated. Our model suggests a possible explanation for the high degree of conservation of the DFG motif: that the flip, modulated by electrostatic changes inherent to the catalytic cycle, allows the kinase to access flexible conformations facilitating nucleotide binding and release.conformational change ͉ DFG motif ͉ imatinib ͉ molecular dynamics simulation ͉ pH dependence

show abstract

Section: Conservation Of the Dfg Motif And Its Role In Kinase Catalysissupporting

confidence: 92%

A conserved protonation-dependent switch controls drug binding in the Abl kinase

Shan

Seeliger

Eastwood

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

239

314

View full text Add to dashboard Cite

show abstract

“…Interconnectivity of the conserved spatial structures sheds light on long-range communication within kinase molecules, which has been observed by numerous authors (13,14,22,24). The unphosphorylated apo-structures are typically the most disorganized: both the R and C spines are broken, and movements of the lobes are not coordinated.…”

Section: Discussionmentioning

confidence: 86%

“…Disassembly of the spine leads to general destabilization of the kinase molecule, which was previously observed in hydrogen-deuterium exchange studies (10, 11) and MD simulations (12). It was demonstrated subsequently that mutation of the spine residues leads to increased flexibility of the activation loop in MAP kinase ERK2 (13) and to a total inactivation of p38␣ MAP kinase (14).Despite the fact that the spine is a conserved feature, present in all active eukaryotic protein kinases, it was not detected earlier as a conserved spatial motif. This is due, in part, to the highly unusual nature of its formation.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

A helix scaffold for the assembly of active protein kinases

Kornev

Taylor

Eyck

2008

Proc. Natl. Acad. Sci. U.S.A.

394

443

View full text Add to dashboard Cite

Structures of set of serine-threonine and tyrosine kinases were investigated by the recently developed bioinformatics tool Local Spatial Patterns (LSP) alignment. We report a set of conserved motifs comprised mostly of hydrophobic residues. These residues are scattered throughout the protein sequence and thus were not previously detected by traditional methods. These motifs traverse the conserved protein kinase core and play integrating and regulatory roles. They are anchored to the F-helix, which acts as an organizing ''hub'' providing precise positioning of the key catalytic and regulatory elements. Consideration of these discovered structures helps to explain previously inexplicable results.graph theory ͉ hydrophobic motifs ͉ structure comparison P rotein kinases represent a large protein superfamily that regulates numerous processes in living cells (1). Malfunction of this regulation typically leads to various diseases, including immunodeficiencies, cancers, and endocrine disorders (2). Multiple sequence alignment identified the most conserved motifs and defined universal subdomains in protein kinases (3). Solving crystal structures of different protein kinases demonstrated not only a conserved core but also the exceptional flexibility of protein kinases. This indicated an important role of dynamics and plasticity for this family (4, 5). Substantial progress has been made in understanding the regulatory mechanisms, although many questions still remain unanswered (6). Recently, we reported a new bioinformatics method that is capable of detecting conserved patterns formed by residues in space without any relation to protein sequence or main chain geometry. Originally, it was created for comparison of protein surfaces (7,8), but later the method was expanded for analysis of the whole molecule and was termed ''Local Spatial Patterns (LSP) alignment'' (9). Application of the method to a set of serine/threonine and tyrosine kinases led to the discovery of an unusual structure, which we termed a ''spine'' (8). The most remarkable feature of the spine is that it is assembled during the protein kinase activation process and provides coordinated movement of the two kinase lobes. In deactivated kinases, the spine is usually broken because of the rearrangement of the C-helix and/or activation loop. Disassembly of the spine leads to general destabilization of the kinase molecule, which was previously observed in hydrogen-deuterium exchange studies (10, 11) and MD simulations (12). It was demonstrated subsequently that mutation of the spine residues leads to increased flexibility of the activation loop in MAP kinase ERK2 (13) and to a total inactivation of p38␣ MAP kinase (14).Despite the fact that the spine is a conserved feature, present in all active eukaryotic protein kinases, it was not detected earlier as a conserved spatial motif. This is due, in part, to the highly unusual nature of its formation. It is comprised of four single residues coming from four different subdomains of the protein kinase molecule (III, IV,...

show abstract

“…2a), we hypothesized that 11 binds into the ATP pocket. Structural ordering of the DFG motif, which forms a part of the magnesium-binding pocket ("DFG-in" state) 24 , is one of the important conformational transitions leading to kinase activation, along with structural changes of the adjacent activation loop. The key difference between SU9516 and 11 is that SU9516 binds to the active DFG-in state of CDK2, whereas our data indicate that 11 may target the inactive state of RIP1.…”

Section: Molecular Modeling Of the Rip1-nec-1 Complexmentioning

confidence: 99%