Defining the conserved internal architecture of a protein kinase

Abstract

Protein kinases constitute a large protein family of important regulators in all eukaryotic cells. All of the protein kinases have a similar bilobal fold, and their key structural features have been well studied. However, the recent discovery of non-contiguous hydrophobic ensembles inside the protein kinase core shed new light on the internal organization of these molecules. Two hydrophobic "spines" traverse both lobes of the protein kinase molecule, providing a firm but flexible connection between its key elements. The spine model introduces a useful framework for analysis of intramolecular communications, molecular dynamics, and drug design.

Figure 1. General architecture of the protein kinase catalytic core

A) Two lobes of a protein kinase molecule (PKA structure 1ATP was used). ATP is bound in a deep cleft between the lobes. The substrate binding groove on the C-lobe is colored sand with the phosphorylation (P) site shown as a yellow sphere. The residues that follow the P-site (P+1 residue) are shown as a dark red surface. B) Five β-strands and a prominent αC-helix from the N-lobe. The G-loop is colored red. C) A large swing motion of the αC-helix is associated with activation (Insulin receptor kinase structures 1IR3 and 1IRK were used). D) Helical content of the C-lobe. The Activation segment is colored red. The primary phosphorylation site, T¹⁹⁷ and its binding partners in PKA are shown as sticks.

Figure 2. The substrate peptide is positioned with respect to the αF-helix by a set of conserved hydrophobic residues in the Activation segment

The phosphorylation site for a PKA substrate is shown as a yellow sphere. The hydrophobic pocket created by the P+1 loop is clearly anchored to the conserved W²²² in the αF-helix via the APE-motif. The P-2 arginine forms a salt bridge with E²³⁰ in the C-terminus of the helix.

Figure 3. Two hydrophobic spines define the internal architecture of every protein kinase

The spines are anchored to the αF-helix and traverse both lobes of the kinase. The C-spine residues are shown as a yellow surface. The R-spine is colored red. The gatekeeper residue is located between the spines (shown as a dark red surface). (Insert: global view of the spines inside the kinase core).

Figure 4. The spines provide a flexible connection between the two lobes of the kinase core

Closed and open conformations of active PKA are shown (PDB IDs 1ATP and 1J3H respectively). The conserved salt bridge between K⁷² and E⁹¹ is formed in the closed configuration and is disrupted in the open state due to the movements in the N-lobe. The spines remain intact during the “breathing” motion of the kinase, which is important for efficient catalysis.