Domain stabilities in protein kinase R (PKR): evidence for weak interdomain interactions

Abstract

PKR (protein kinase R) is induced by interferon and is a key component of the innate immunity antiviral pathway. Upon binding dsRNA, PKR undergoes autophosphorylation reactions that activate the kinase, leading it to phosphorylate eIF2alpha, thus inhibiting protein synthesis in virally infected cells. PKR contains a dsRNA-binding domain (dsRBD) and a kinase domain. The dsRBD is composed of two tandem dsRNA-binding motifs. An autoinhibition model for PKR has been proposed, whereby dsRNA binding activates the enzyme by inducing a conformational change that relieves the latent enzyme of the inhibition that is mediated by the interaction of the dsRBD with the kinase. However, recent biophysical data support an open conformation for the latent enzyme, where activation is mediated by dimerization of PKR induced upon binding dsRNA. We have probed the importance of interdomain contacts by comparing the relative stabilities of isolated domains with the same domain in the context of the intact enzyme using equilibrium chemical denaturation experiments. The two dsRNA-binding motifs fold independently, with the C-terminal motif exhibiting greater stability. The kinase domain is stabilized by about 1.5 kcal/mol in the context of the holenzyme, and we detect low-affinity binding of the kinase and dsRBD constructs in solution, indicating that these domains interact weakly. Limited proteolysis measurements confirm the expected domain boundaries and reveal that the activation loop in the kinase is accessible to cleavage and unstructured. Autophosphorylation induces a conformation change that blocks proteolysis of the activation loop.

Figure 1

PKR structure. A: Schematic representation of PKR domain organization. The letters “A,B,C” refer to the principal trypsin cleavage sites. The grey portion of the kinase domain corresponds to the kinase insert region. B: Structure of PKR kinase domain. The protein backbone is shown as a ribbon diagram in blue with the activation loop in brown. The three tryptophan side chains are shown in Van der Waals representation in red and AMPPNP is drawn in stick format. The solvent accessible surface is beige. The coordinates (PDB 2A19) were obtained from the crystal structure of a complex of the PKR kinase domain with eIF2α and AMPPNP (11) and the figure was rendered with PYMOL (Delano Scientific, Palo Alto, CA).

Figure 2

Limited proteolysis of PKR. PKR was incubated at 1 mg/ml at several ratios of PKR: trypsin at 20 °C for 30 minutes. Reactions were quenched by the addition of SDS sample loading buffer and heating to 90°C for 10 minutes. The samples were run on a 4–12% acrylamide bis-tris gel under denaturing conditions and visualized with Coomassie blue staining. Lane 1 contains unphosphorylated PKR that has not been digested. Lanes 2–5 contain unphosphorylated PKR with increasing concentrations of trypsin. Lanes 6–9 contain phosphorylated PKR and increasing concentrations of trypsin.

Figure 3

Analysis of the stability of dsRBM1 and dsRBD by equilibrium urea unfolding titrations. Samples were prepared at 0.1 mg/ml (dsRBM1) or 0.15 mg/ml (dsRBD) and incubated at variable urea concentrations at 20°C for 3 hours. CD data were collected at 222 nm in a 5 mm path length cuvette. The data were background-subtracted, normalized and fit with a two-state model (dsRBM1) or a three-state model (dsRBD) using the program SAVUKA. The points are the normalized data for dsRBM1 () and dsRBD (). and the lines are the respective two- and three-state fits. The inset shows the residuals. The best-fit parameters are in Table 1.

Figure 4

Analysis of the stability of the kinase domain. Samples were prepared at 0.15 mg/ml and variable urea concentrations and incubated at 20°C for 3 hours. Unfolding was monitored using CD at 222 nm () and tryptophan fluorescence emission with excitation at 295 and emission at 340 nm (). A) Raw data. B) Background subtracted and normalized data. The lines are the global fit of the CD and fluorescence data to a three-state model obtained using SAVUKA.

Figure 5

Analysis of PKR stability. Full length PKR was prepared at 0.15 mg/ml and variable urea concentrations and incubated at 20°C for 3 hours. Unfolding was monitored using CD at 222 nm () and tryptophan fluorescence emission with excitation at 295 and emission at 340 nm (). A) Raw data. B) Background subtracted and normalized data. The lines are the global fit of the CD and fluorescence data to a three-state model obtained using SAVUKA.

Figure 6

Analysis of the stability of phosphorylated PKR stability. Full length PKR was prepared at 0.15 mg/ml and variable urea concentrations and incubated at 20°C for 3 hours. Unfolding was monitored using CD at 222 nm () and tryptophan fluorescence emission with excitation at 295 and emission at 340 nm (). A) Raw data. B) Background subtracted and normalized data. The lines are the global fit of the CD and fluorescence data to a three-state model obtained using SAVUKA.

Figure 7

Sedimentation velocity analysis of the interaction of dsRBD and kinase domains. A) Continuous sedimentation coefficient distribution analysis of dsRBD (black), kinase domain (blue) and 1:1 (molar) mixture of dsRBD and kinase domain (red). The distributions are normalized by peak height and offset for clarity. The small feature near 4 S in the samples containing kinase domain is assigned as an irreversible kinase dimer contaminant. Conditions: sample concentrations, 0.5 mg/ml (dsRBD), 0.5 mg/ml (kinase domain), 1 mg/ml (mixture); rotor speed, 55,000 RPM; temperature, 20°C; interference optics. B) g(s*) analysis of 1:1 mixtures of dsRBD: kinase domain at concentrations of 3.2 mg/ml (red), 1.1 mg/ml (blue) and 0.36 mg/ml (black). The distributions are normalized by concentration. Conditions: rotor speed, 55,000 RPM; temperature, 20°C; interference optics.