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. 2016 Jan 19;55(2):253-61.
doi: 10.1021/acs.biochem.5b01171. Epub 2015 Dec 30.

Role of the Interdomain Linker in RNA-Activated Protein Kinase Activation

Bushra Husain  1 , Christopher Mayo  1 , James L Cole  1
Affiliations

Role of the Interdomain Linker in RNA-Activated Protein Kinase Activation

Bushra Husain et al. Biochemistry. .

Abstract

RNA-activated protein kinase (PKR) is a key component of the interferon-induced antiviral pathway in higher eukaryotes. Upon recognition of viral dsRNA, PKR is activated via dimerization and autophosphorylation. PKR contains two N-terminal dsRNA binding domains (dsRBD) and a C-terminal kinase domain. The dsRBDs and the kinase are separated by a long, unstructured ∼80-amino acid linker in the human enzyme. The length of the N-terminal portion of the linker varies among PKR sequences, and it is completely absent in one ortholog. Here, we characterize the effects of deleting the variable region from the human enzyme to produce PKRΔV. The linker deletion results in quantitative but not qualitative changes in catalytic activity, RNA binding, and conformation. PKRΔV is somewhat more active and exhibits more cooperative RNA binding. As we previously observed for the full-length enzyme, PKRΔV is flexible in solution and adopts a range of compact and extended conformations. The conformational ensemble is biased toward compact states that might be related to weak interactions between the dsRBD and kinase domains. PKR retains RNA-induced autophosphorylation upon complete removal of the linker, indicating that the C-terminal, basic region is also not required for activity.

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Figures

Figure 1
Figure 1
PKR domains and linker regions. (a) Domains of PKR and linker deleted constructs. The interdomain linker contains a variable region (169–229) and a basic region (230–252) depicted as blue and red solid lines respectively. (b) Linker sequence alignment for PKR orthologs prepared using SeaView. (c) Prediction of disordered regions in hPKR by PONDR. The region indicated by the red line (191–253) is predicted to be disordered. The entire linker (169–252) is indicated by the black line.
Figure 2
Figure 2
Effect of the variable linker region on PKR activation. (a) dsRNA-independent activation of hPKR and PKRΔV. (b) dsRNA-induced activation of PKRΔV. Activation was monitored at 0.1 µM PKRΔV with varying concentrations of 20 bp (black), 30 (blue) and 40 bp (red) dsRNA. The data are normalized relative to the extent of (wild-type) hPKR activation at 0.03 uM 40 bp dsRNA.
Figure 3
Figure 3
Dimerization of PKRΔV. The Kd for self-association of PKRΔV was measured using sedimentation equilibrium. Data (open circles) were collected for 0.25, 0.5, 1, and 2 mg/mL PKRΔV at two rotor speeds: 18,000 rpm (blue) and 22,000 rpm (red). For clarity, only every other data point is shown and the curves are vertically offset. The solid lines indicate a global fit of the data to a monomer-dimer equilibrium model. The best-fit Kd = 1.105 (1.103, 1.210) mM with an rmsd of 0.0256 fringes Inset: residuals.
Figure 4
Figure 4
Effect of the variable linker region on RNA binding. PKRΔV binding to 40 bp dsRNA measured by sedimentation velocity. Data were obtained at 0.75 uM RNA and variable protein concentrations. (a) Normalized g(s*) distributions for samples containing 0 (black), 0.5 (blue), 1 (red), 3 (green) and 6 (tan) equivalents of PKRΔV. (b) Global analysis of difference curves for PKRΔV binding to 40 bp dsRNA using a model of three sequential binding events. The top panels show the data (points) and fit (lines) and the bottom panels show the residuals (points). The best fit parameters are presented in Table 1.
Figure 5
Figure 5
SAXS analysis of PKRΔV conformation. Data were collected at 4°C at a protein concentration of 5 mg/ml. (a) Scattering data (points) and EOM fit (solid red line). Inset: Guinier analysis yields Rg = 33.3 ± 0.2 Å. (b) p(r) distance distribution produced by transforming the data in part (a) using GNOM. Rg (c) and Dmax (d) distributions produced by fitting the data in part (a) using EOM., The distributions of the random pool of 10,000 structures are shown in black dotted lines and the distributions selected by the genetic algorithm to fit the experimental data are shown in solid blue. The error bars correspond to the standard deviations of the distributions produced from three runs of GAJOE.
Figure 6
Figure 6
SAXS analysis of PKRΔVB conformation. Data were collected at 4°C at a protein concentration of 5 mg/ml. (a) Scattering data (points) and BUNCH fit (solid red line). Inset: Guinier analysis yields Rg = 30.5 ± 0.3 Å. (b) p(r) distance distribution produced by transforming the data in part (a) using GNOM. (c) Structural model produced by BUNCH. The domains are shown in surface representation: dsRBD1 (orange), dsRBD2 (cyan) and kinase (salmon). The linker residues are depicted as yellow spheres.
Scheme 1
Scheme 1
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