Structural Basis of Protein Kinase R Autophosphorylation

Abstract

The RNA-activated protein kinase, PKR, is a key mediator of the innate immunity response to viral infection. Viral double-stranded RNAs induce PKR dimerization and autophosphorylation. The PKR kinase domain forms a back-to-back dimer. However, intermolecular ( trans) autophosphorylation is not feasible in this arrangement. We have obtained PKR kinase structures that resolves this dilemma. The kinase protomers interact via the known back-to-back interface as well as a front-to-front interface that is formed by exchange of activation segments. Mutational analysis of the front-to-front interface support a functional role in PKR activation. Molecular dynamics simulations reveal that the activation segment is highly dynamic in the front-to-front dimer and can adopt conformations conducive to phosphoryl transfer. We propose a mechanism where back-to-back dimerization induces a conformational change that activates PKR to phosphorylate a "substrate" kinase docked in a front-to-front geometry. This mechanism may be relevant to related kinases that phosphorylate the eukaryotic initiation factor eIF2α.

Figure 1.. Arrangement of PKR kinase monomers in two crystal forms.

The top panels show a surface representation and the bottom panels show a cartoon representation. The alternating interfaces form a continuous, filament-like assembly within the crystal lattices. For clarity, only six protomers are shown in surface representation and three are shown in cartoon representation to illustrate the unique interfaces. A) Apo crystal form. B) AMPPNP complex crystal form. One protein chain occupies the asymmetric unit (AU) in the apo crystal form. Three protein chains, labeled A, B, and C, are in the AU in the AMPPNP complex. The face-to-face (FTF) and back-to-back (BTB) interfaces are indicated in the cartoon representation of each structure. Each of the FTF interfaces involves exchange of activation segments except for the C-C chains, la.

Figure 2.. Structural comparison of unphosphorylated and phosphorylated PKR kinase.

A) Alignment of the three unique protomers present in the asymmetric unit of the AMPPNP complex of the unphosphorylated PKR kinase domain with the AMPPNP complex of a phosphorylated PKR kinase domain (PDB 2A19, chain B). The color scheme is indicated in the legend. B) Comparison of the active sites. For clarity, only chain B of the unphosphorylated AMPPNP complex is shown. The nucleotide, free phosphate, and important side chains are rendered as sticks. The Mg²⁺ is indicated as a sphere. Hydrogen bond and salt-bridge interactions in the unphosphorylated kinase are denoted as dotted lines. The R-spine is shown in surface representation. A superposition of all three chains of the unphosphorylated enzyme with phosphorylated PKR kinase domain is shown in Figure S2.

Figure 3.. Analysis of the PKR kinase face-to-face dimer with exchange of activation loops.

A) Structure of the interface. The A and B chains of the AMPPNP complex of PKR kinase are depicted using the color scheme from Figure 1. The protomers are indicated in cartoon representation with the disordered regions of the activation loop and the C-terminus shown as dashes. The bound nucleotide is depicted in stick representation. B) Detailed view of the interactions stabilizing the interface. Key side chain and main chain atoms are rendered as sticks. Hydrogen bond and salt-bridge interactions are denoted by dashed lines. G466 is shown as a sphere. C) Structural alignment of a monomeric, phosphorylated PKR kinase (2A19) onto chain B forming a domain-swapped FTF dimer with chain A. The side chain and main chain atoms involved in polar interactions at the interface are rendered as sticks. D) Effect of interface mutations on PKR activation. The PKR autophosphorylation activity was assayed as a function of dsRNA concentration. The data are normalized to the maximal activation of wild-type PKR.

Figure 4.. Structure of the PKR kinase face-to-face dimer without exchange.

Two symmetry-related C chains of the AMPPNP complex of PKR kinase forming a FTF dimer without exchange of activation segments are depicted using the color scheme from Figure 1. The chains are referred to as C and Cʹ. A) Comparison of the FTF interfaces. The A:B dimer with exchange and the C:Cʹ dimer without exchange were aligned on the A and C protomers on the left, treating the dimers as rigid units. Relative to the Cʹ protomer, the B protomer is rotated by 38°. The bound nucleotide in chain C is depicted in stick representation. B) Detailed view of the interactions stabilizing the interface. The orientation corresponds to a 90° rotation of the structure depicted in part A. Key side chain and main chain atoms are rendered as sticks. Hydrogen bond and salt-bridge interactions are denoted by dashed lines.

Figure 5.. Molecular dynamics analysis of the face-to-face dimer with exchange of activation loops.

A) RMSD over the course of the 1 μs simulation relative to the initial structure for subunit A (top) and subunit B (bottom). The highly dynamic β4-β5 loop and activation segment were excluded. B) Distribution of the center-of-mass distances between monomers for the FTF dimer with exchange. The inset shows the trajectory of the distances over the simulation. C) Two-dimensional histogram of the intermonomer T446-ATP and T446-D414 distances. The data correspond to the protomer A Oγ (T446)-protomer B Oγ (ATP) and protomer A Oγ (T446)-protomer B Oδ (D414) distances. The closest distances were recorded for the groups of Oγ (ATP) and the Oδ (D414) atoms. The rainbow color scale for the 1 Å ×1 Å pixels corresponds to occupancies from 1 (red) to 200 (purple). D) Structure of the active site of protomer A corresponding to the pixel circled in part C. The intermonomer T446-ATP and T446-D414 distances are 5.96 Å and 4.45 Å, respectively. The Oγ (T446)-Pγ (ATP)-Pβ (ATP) pseudo-angle is 143.5°.

Figure 7.. Multistep model for PKR activation.

The kinase domain of monomeric PKR exists in an inactive conformation. In the first step, PKR binds to activating RNAs via the tandem dsRBDs (dsRBD1 and dsRBD2), bringing two kinase domains into proximity to promote dimerization. Formation of the BTB dimer stabilizes the prone-to autophosphorylate-conformation. In the second step, the BTB dimer phosphorylates the activation loop of a PKR monomer docked in a domain-swapped, FTF geometry. The kinase domain in the inactive conformation is depicted in blue and the prone-to-autophosphorylate and active conformations are shown in green.