A helix scaffold for the assembly of active protein kinases

Abstract

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,...

Identification of a hidden strain switch provides clues to an ancient structural mechanism in protein kinases

“…A regulatory role for the strain is also supported by recent NMR studies on PKA, which show that ATP binding induces global conformational changes coupling the ATP and substrate binding lobes (12,13). This coupling has been suggested to involve the hydrophobic spine connecting the ATP and substrate-binding lobes (16,17). Our analysis suggests that in addition to the hydrophobic spine, switching of the HRD backbone from relaxed to strained conformation may also contribute to allosteric coupling between ATP and substrate binding lobes (Fig.…”

“…More recently, a network of hydrophobic interactions connecting the ATP and substrate binding lobes of the kinase domain, called the regulatory spine, has been suggested to play an important role in kinase activation (16,17). According to the spine model, the assembly of the hydrophobic interactions linking the ATP and substrate binding regions leads to kinase activation (16,17).…”

Dynamic architecture of a protein kinase

“…In contrast, the catalytic spine (C-spine) is completed by the binding of the adenine ring of ATP. Both spines are anchored to the hydrophobic αF-helix (9). Although these analyses helped to define important structural elements and organizing principles for EPKs, many long-distance allosteric effects of mutations and of ligand and protein binding remained poorly understood.…”

Mutation of a kinase allosteric node uncouples dynamics linked to phosphotransfer

“…1). The assembled regulatory "R spine" is the hallmark signature of an active kinase, and the disassembled-assembled R-spine switch constitutes the dynamic link between kinase inactivation-activation (5). The catalytic "C spine" is completed by binding of the ATP adenine ring that primes the kinase for catalysis (5,6).…”