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. 2006 Nov 21;103(47):17783-8.
doi: 10.1073/pnas.0607656103. Epub 2006 Nov 9.

Surface comparison of active and inactive protein kinases identifies a conserved activation mechanism

Alexandr P Kornev  1 , Nina M Haste, Susan S Taylor, Lynn F Ten Eyck
Affiliations

Surface comparison of active and inactive protein kinases identifies a conserved activation mechanism

Alexandr P Kornev et al. Proc Natl Acad Sci U S A. .

Abstract

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 Asp-Phe-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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Surface comparison of two active protein kinases (PKA and Cdk2) is capable to detect the most conserved, functionally important residues. (A) Diagram of known interactions between the protein kinase catalytic core, ATP and a substrate (PKA numbering is used). Red arrow indicates catalyzed transfer of the ATP γ-phosphate to hydroxyl group of a protein substrate. Catalytically important residues which are in contact with ATP and/or substrate are shaded yellow. Secondary structures and residues which are known to be involved in regulation of the catalytic activity are shaded gray. Hydrophobic interactions between the HRD motif, the DFG motif and the αC helix are shown by black arrows. The important polar contacts are shown by dashed lines. (B) Two graphs representing surface similarity between PKA and Cdk2. To indicate positions of the detected residues in protein kinase sequence, we color-coded the vertices according to the Hanks and Hunter (24) designation for subdomains. The locations of the subdomains are shown as color-coded ribbons. (Inset) Close-up of the highly interconnected DFG motif residues (blue vertices).
Fig. 2.
Fig. 2.
Total number of connections detected for different PKA residues on similarity graphs after comparison of active PKA structure to sets of active and inactive protein kinases. The open bars show the AA score, from comparison of active PKA to other active protein kinases (see text). The filled bars show the AI score, from comparison of active PKA to a set of inactive protein kinases. The list of compared structures is presented in Table 1.
Fig. 3.
Fig. 3.
Proposed model for the correlation between phosphorylation of the activation loop and the active configuration of the DFG motif. (A) DFG -glycine moves out its amide nitrogen upon inactivation breaking a conserved hydrogen bond to the DFG aspartate (dashed red line). Cdk2 structures were used for the illustration (magenta, active; cyan, inactive; see Table 1). (B) Alignment (main chain atoms only) of the magnesium-binding loops of 23 active kinases (Table 2) demonstrates conserved geometry for this part of the activation segment. Two conserved hydrogen bonds are shown as red arrows. One is between DFG aspartate and the DFG glycine; the other is a 3-turn between the DFG phenylalanine and the DFG + 2 residue. (C) A cascade of conserved hydrogen bonds connecting the phosphorylation site of PKA and the catalytically important DFG aspartic acid (I–IV). Carbons of the activation loop are colored white; carbons of the catalytic loop are colored tan; carbons of the β7–β8 sheet, which precedes the DFG motif, are colored green. The six Cα atoms of the magnesium-binding loop are shown as spheres, the immobilized atoms are colored tan, and the rest are colored yellow. Two peptide bonds, which are able to rotate around Cα atoms, are shown as blue planes.
Fig. 4.
Fig. 4.
Mechanism of Cdk2 partial activation induced by the cyclin binding. The cyclin ribbon and its residue labels are colored cyan; Cdk2 ribbons and residues are colored green. Cyclin is involved in configuring the magnesium-binding loop through a set of hydrogen bonds to R150 (K189 in PKA). In addition, the PSTAIRE arginine (R50) is coordinated by cyclin binding to main chain of the DFG residue. The absence of negatively charged phosphate is compensated by a glutamic acid residue (E162).
Fig. 5.
Fig. 5.
Hydrophobic “spine” assembled in active kinases consists of four hydrophobic residues corresponding to four PKA residues L95, L106, Y164 and F185. (A) Alignment of hydrophobic residues which form the spine in 23 active eukaryotic and prokaryotic kinases (Table 2). (B) Position of the spine in PKA. The formation is shown as a red Connolly surface with probe radius of 1.4 Å. Blue spheres represent Cα atoms of the residues that lie along the “rocking” motion axis detected in molecular dynamics simulation (22).
Fig. 6.
Fig. 6.
General model of protein kinase activation (PKA example). (A) Active conformation. Phosphothreonine T197 arranges the magnesium-binding loop positioning the DFG aspartate for interaction with the ATP and the DFG phenylalanine for building up the hydrophobic “spine.” The αC helix flips to complete the spine formation and simultaneously secures it with the K72–E91 polar contact. The spine residues are shown as blue disks and the shaded gray portion of the N-lobe. The spine stabilizes the protein kinase molecule, which can perform coordinated motions going through open (Left) and closed (Right) conformations during the catalytic cycle. (B) Inactive conformation. The magnesium-binding loop and the spine are distorted, destabilizing the molecule. The lobes move independently. The unconstrained magnesium-binding loop becomes flexible and can attain different inactive configurations.
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