A conserved Glu-Arg salt bridge connects coevolved motifs that define the eukaryotic protein kinase fold

Abstract

Eukaryotic protein kinases (EPKs) feature two coevolved structural segments, the Activation segment, which starts with the Asp-Phe-Gly (DFG) and ends with the Ala-Pro-Glu (APE) motifs, and the helical GHI subdomain that comprises αG-αH-αI helices. Eukaryotic-like kinases have a much shorter Activation segment and lack the GHI subdomain. They thus lack the conserved salt bridge interaction between the APE Glu and an Arg from the GHI subdomain, a hallmark signature of EPKs. Although the conservation of this salt bridge in EPKs is well known and its implication in diseases has been illustrated by polymorphism analysis, its function has not been carefully studied. In this work, we use murine cAMP-dependent protein kinase (protein kinase A) as the model enzyme (Glu208 and Arg280) to examine the role of these two residues. We showed that Ala replacement of either residue caused a 40- to 120-fold decrease in catalytic efficiency of the enzyme due to an increase in K(m)(ATP) and a decrease in k(cat). Crystal structures, as well as solution studies, also demonstrate that this ion pair contributes to the hydrophobic network and stability of the enzyme. We show that mutation of either Glu or Arg to Ala renders both mutant proteins less effective substrates for upstream kinase phosphoinositide-dependent kinase 1. We propose that the Glu208-Arg280 pair serves as a center hub of connectivity between these two structurally conserved elements in EPKs. Mutations of either residue disrupt communication not only between the two segments but also within the rest of the molecule, leading to altered catalytic activity and enzyme regulation.

Figure 1. The Activation segment and the GHI subdomain in ELKs

A) Comparison of eukaryotic protein kinase A (PKA) and eukaryote-like kinase Aminoglycoside phosphotransferase (Aph). The N-lobe is colored white. The three helixes from the C-lobe that are common for both EPKs and ELKs are colored tan. Other helixes in the C-lobe are semitransparent and colored yellow. The Activation segment in EPKs and the corresponding part of the C-lobe in ELKs are shown as red ribbon. B) The Glu208-Arg280 salt bridge connects the Activation segment (red ribbon) and the αH-αI loopin the GHI subdomain (yellow helices). C)Conserved hydrophobic interactions dock the Activation segment and the GHI-subdomain to the αF-helix (colored tan), the central structural element of the C-lobe. Trp222 from the αF-helix is shown as white surface. Hydrophobic residues from the Activation segment are shown as red surface. Hydrophobic residues from the GHI-subdomain are shown as sand surface. D) Extended Activation segment. The Mg-binding loop with the DFG-motif is colored bright red. The Activation loop with the primary phosphorylation site is colored dark red. The P+1 loop is colored yellow. The APE-motif is colored teal. The additional APE-F linker is shown as white ribbon.

Figure 2. Mutations caused increased flexibility in protein structure

Average B-factor of the small lobe (2, grey), large lobe (3, tan), glycine-rich loop (4, yellow) and the αH-αI loop (5, red) was calculated using CNS_Solve 1.2, and compared to the average B-factor of the corresponding whole molecule (1, black). Residues are colored by atom types (N, blue, O, red, C, yellow). The zoom view shows the electron density map of the αH-αI loop region ²⁷⁵VDLTKRFNLKN²⁸⁶ in C^R280A (top) and C^E208A (bottom) structures. Electron density map of the same region in the wild type structure is very similar to that of C^E208A. Two segments (left, residues 274–279, right, residues 279–286) were shown for a better view. Side chains of most residues in this region except Phe281 were disordered (A in the parenthesis indicated that the residue was replaced by Ala in the refinement). 2Fo-Fc electron density map was contoured at 1σ level.

Figure 3

A) Comparison of the Glu/Ala208 and Arg/Ala280 sites in C^E208A (right), C^R280A (left) and the wild type C-subunit (center). The turquoise spheres represent the water molecules that occupy void space caused by the mutations. The color scheme for the stick representation of residues is by atom types; N, blue, O, red, C, grey). Connolly surface was calculated from Leu277, Ala218 and the aliphatic parts of Glu208, Asp283, Glu274 and Arg280, and shown in 40% transparency colored in light grey. B) Hydrophobic environment of Glu208-Arg280 pair is highly conserved. Left, PKA (PDB ID 1L3R), right, CDK2 (PDB ID 1FIN). In PKA Leu277 from the αH-αI loop and Ala218 from the αF-helix caps the ion-pair of Glu208-Arg280. In CDK2, Pro271 and Ala183 achieve the same function to shield its Glu-Arg ion pair from solvent. C) Solvent accessible surface area (ASA) of Glu208 and Arg280 compared to selected surface exposed Glu or Arg residues in PKA. ASA was calculated on the PISA server, using a probe of 1.6Å. ASA values of two fully solvent exposed arginines, Arg194 and Arg256 were averaged as comparison. Similarly, ASA of two fully solvent exposed glutamic acids, Glu248 and Glu331 were also averaged for comparison.

Figure 4. Comparison of hydrogen deuterium exchange profiles of C^E208A, C^R280A and the wild type C-subunit

Crystal structure of the wild type C-subunit is rendered in cartoon representation with the N-lobe colored white and the C-lobe colored tan. Regions that exhibit increased H/D exchange in the mutants are colored red. Comparison of the time course of H/D exchange profile for four peptides in C^E208A ( triangle), C^R280A (reversed triangle) and wild type C-subunit (circle) are shown. Time points are indicated.

Figure 5. Decreased folding stability of C^E208A and C^R280A

Urea-denaturation curves for WT, C^E208A and C^R280A were compared. The fraction of protein unfolded is shown as a function of urea concentration as measured by intrinsic tryptophan fluorescence.

Figure 6. Glu208 or Arg280 is essential for phosphorylation of PKA by PDK1

A) C-subunit mutants were expressed overnight at 16°C, either alone or with PDK1, in E. coli, and the soluble fraction of the cell lysates were immunoblotted for phospho-Thr197 phosphorylation, C-subunit, and PDK1. B) Purified PDK1 was incubated with purified mutants His(6)-C^R194A, His(6)-C^R194A/E208A, and His(6)-C^R194A/R280A and Mg-ATP for 90 min at room temperature and immunoblotted for phospho-Thr197 phosphorylation and C-subunit. Untagged wild type C-subunit was loaded as an antibody control.