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.

Fig. 1.

LSP alignment of active conformation of PKA to active conformations of other kinase. Four different families were used in the comparison: AGC kinases (PKC and ROCK1), CMGC kinases (CDK2 and SKY1), calcium/calmodulin kinases (PHK and DAPK), and tyrosine kinases (SRC and IRK). Residues that constitute the two conserved spines are spread along the kinase sequence: R spine residues are marked as red dots; yellow dots mark the C spine residues. Highly scored αF-helix is marked by red square.

Fig. 2.

R and C spines flank the F-helix and span the protein kinase core. The PKA structure is shown as a prototype. (A and B) The hydrophobic part of the R spine is shown as a red molecular surface. The C spine is colored yellow. The adenine ring of ATP completes the C spine. (C) Eight hydrophobic residues that constitute the C spine in 22 active kinases from six different groups (see Table S2 for the list of kinases).

Fig. 3.

αH-helix anchoring structure in PKA. Molecular surfaces of the highly scored residues are shown: olive, from the αF-helix, tan, from the αH-helix. L²⁷³ fits as a “knob” into a “hole” formed by conserved W²²², G²²⁵, and Y²²⁹.

Fig. 4.

Catalytic and activation loop anchoring in PKA. (A) N- and C-terminal parts of the catalytic loop are secured by the spines. The middle part is docked to A²²³ and L²²⁴ from the αF-helix. Three catalytically active residues are shown: D¹⁶⁶, K¹⁶⁸, and N¹⁷¹. (B and C) Activation loop anchoring. Its N terminus binds to the β₈-strand that makes both polar and hydrophobic bonds to the β₇-strand from the C spine. The β₈-strand is also bound to the αF-helix via hydrophobic interaction with the conserved I¹⁵⁰. This positions the N terminus of catalytically active D184, whereas its C terminus is secured by the R spine.

Fig. 5.

Organization of the substrate-binding network in PKA. (A) V²²⁶ stabilizes Y²⁰⁴, which anchors the activation segment (colored bright red) and orients side chain of K¹⁶⁸. E²³⁰ interacts with the substrate arginine. (B) W²²² serves as a docking platform for the APE motif, which serves as a foundation for the P + 1 loop. (C) Closeup of the APE-motif interactions. R²⁸⁰ forms multiple hydrogen bonds to the αF-helix, the APE motif, and the αH-αI loop. (D) General chart of the YLAPEL motif that mediates interaction between the αF-helix, substrate binding, and docking sites.