Eukaryotic protein kinases evolved as a family of highly dynamic molecules with strictly organized internal architecture. A single hydrophobic F-helix serves as a central scaffold for assembly of the entire molecule. Two non-consecutive hydrophobic structures termed “Spines” anchor all the elements important for catalysis to the F-helix. They make firm, but flexible, connections within the molecule providing a high level of internal dynamics of the protein kinase. During the course of evolution, protein kinases developed a universal regulatory mechanism associated with a large Activation Segment that can be dynamically folded and unfolded in the course of cell functioning. Protein kinases thus represent a unique, highly dynamic and precisely regulated set of switches that control most biological events in eukaryotic cells.
The surface comparison of different serine-threonine and tyrosine kinases reveals a set of 30 residues whose spatial positions are highly conserved. The comparison between active and inactive conformations identified the residues whose positions are the most sensitive to activation. Based on these results, we propose a model of protein kinase activation. This model explains how the presence of a phosphate group in the activation loop determines the position of the catalytically important aspartate in the AspPhe-Gly motif. According to the model, the most important feature of the activation is a ''spine'' formation that is dynamically assembled in all active kinases. The spine is comprised of four hydrophobic residues that we detected in a set of 23 eukaryotic and prokaryotic kinases. It spans the molecule and plays a coordinating role in activated kinases. The spine is disordered in the inactive kinases and can explain how stabilization of the whole molecule is achieved upon phosphorylation.protein surface ͉ graph theory
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,...
Protein kinases are dynamic molecular switches that have evolved to be only transiently activated. Kinase activity is embedded within a conserved kinase core, which is typically regulated by associated domains, linkers and interacting proteins. Moreover, protein kinases are often tethered to large macromolecular complexes to provide tighter spatiotemporal control. Thus, structural characterization of kinase domains alone is insufficient to explain protein kinase function and regulation in vivo. Recent progress in structural characterization of cyclic AMP-dependent protein kinase (PKA) exemplifies how our knowledge of kinase signalling has evolved by shifting the focus of structural studies from single kinase subunits to macromolecular complexes.
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