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. 2009 Apr 10;387(4):910-20.
doi: 10.1016/j.jmb.2009.02.019. Epub 2009 Feb 14.

Analysis of PKR structure by small-angle scattering

Jennifer VanOudenhove  1 , Eric Anderson, Susan Krueger, James L Cole
Affiliations

Analysis of PKR structure by small-angle scattering

Jennifer VanOudenhove et al. J Mol Biol. .

Abstract

Protein kinase R (PKR) is a key component of the interferon antiviral defense pathway. Upon binding double-stranded RNA, PKR undergoes autophosphorylation reactions that activate the kinase. PKR contains an N-terminal double-stranded RNA binding domain, which consists of two tandem double-stranded RNA binding motifs, and a C-terminal kinase domain. We have used small-angle X-ray scattering and small-angle neutron scattering to define the conformation of latent PKR in solution. Guinier analysis indicates a radius of gyration of about 35 A. The p(r) distance distribution function exhibits a peak near 30 A, with a broad shoulder extending to longer distances. Good fits to the scattering data require models that incorporate multiple compact and extended conformations of the two interdomain linker regions. Thus, PKR belongs to the growing family of proteins that contain intrinsically unstructured regions. We propose that the flexible linkers may allow PKR to productively dimerize upon interaction with RNA activators that have diverse structures.

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Figures

Figure 1
Figure 1
Sequence alignment of PKR linker. The sequence of the second linker region in human PKR (residues 169–257) was aligned with the corresponding regions in R. norvegicus and M. auratus enzymes. The residues are colored as follows: acidic, blue; basic, red; polar uncharged, green; nonpolar, grey.
Figure 2
Figure 2
Small angle scattering analysis of PKR. Protein concentrations: 1 mg/mL (blue), 2 mg/mL (red) and 4 mg/mL (black). A) SAXS scattering profiles. B) SAXS Guinier plots. The solid lines indicated regions used in the Guinier fits. C) SAXS p(r) distributions. D) SANS scattering profiles. E) SANS Guinier plots. The solid lines indicated the regions used in the Guinier fits. F) SANS p(r) distributions. Errors in A and D correspond to 1 stand deviation. Errors in B and C are not shown for clarity and are within the scatter of the data.
Figure 3
Figure 3
EOM analysis of PKR SAXS data. A) Scattering profile. The red points are the experimental data (2mg/mL) and the solid black line is the fit. Error bars are omitted for clarity and correspond to those shown in figure 2A. For details see Materials and Methods. B) Rg distribution. The red line is the pool and the blue line corresponds to the ensemble of structures selected by the genetic algorithm. The same color convention is used for B–F. C) Dmax distribution. D) Distribution of dsRBM1 - dsRBM2 distances. E) Distribution of dsRBM1 - kinase distances. F) Distribution of dsRBM2 - kinase distances.
Figure 4
Figure 4
Overlay of PKR structures selected by EOM. The 20 PKR conformations from the ensemble selected by EOM analysis of the 2 mg/mL SAXS data are superimposed by alignment on the kinase domain. The kinase domain is shown in a tan surface representation, the dsRBM1 and dsRBM2 are shown in ribbon representation and the Cα atoms in the flexible linkers are shown as spheres. Each of the 20 conformations is depicted in a different color. In the view on the left, the kinase domain is oriented with the C-lobe on the left and the N-lobe to the right. The arrows indicate representative compact (red) and extended (magenta) conformers. The ensemble on the right is rotated by −90° about the y-axis such that the kinase N-lobe points out of the page.
Figure 5
Figure 5
Overlay of structures generated by simulated annealing runs using BUNCH. The five PKR conformations from BUNCH analysis of the 2 mg/mL SAXS data are superimposed by alignment on the kinase domain. The kinase domain is shown in a tan surface representation, the dsRBM1 and dsRBM2 are shown in ribbon representation and the Cα atoms in the flexible linkers are shown as spheres. The orientation corresponds to the right-hand representation in figure 4.
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