The structure of the PERK kinase domain suggests the mechanism for its activation

Abstract

The endoplasmic reticulum (ER) unfolded protein response (UPR) is comprised of several intracellular signaling pathways that alleviate ER stress. The ER-localized transmembrane kinase PERK is one of three major ER stress transducers. Oligomerization of PERK's N-terminal ER luminal domain by ER stress promotes PERK trans-autophosphorylation of the C-terminal cytoplasmic kinase domain at multiple residues including Thr980 on the kinase activation loop. Activated PERK phosphorylates Ser51 of the α-subunit of translation initiation factor 2 (eIF2α), which inhibits initiation of protein synthesis and reduces the load of unfolded proteins entering the ER. The crystal structure of PERK's kinase domain has been determined to 2.8 Å resolution. The structure resembles the back-to-back dimer observed in the related eIF2α kinase PKR. Phosphorylation of Thr980 stabilizes both the activation loop and helix αG in the C-terminal lobe, preparing the latter for eIF2α binding. The structure suggests conservation in the mode of activation of eIF2α kinases and is consistent with a `line-up' model for PERK activation triggered by oligomerization of its luminal domain.

Figure 1

Overall structure of the mPERK KD dimer. Ribbon drawings of two mPERK KD protomers are colored cyan and green, respectively. mPERK KD dimerizes through its N-terminal lobe. Thr980 residues are phosphorylated in this dimer structure and are shown as ball-and-stick models. Regions missing from the electron-density map are indicated by dotted lines.

Figure 2

Structural comparisons between mPERK KD and PKR KD. (a) Superimposition of the mPERK KD N-lobe (gold) with the active PKR KD N-lobe (silver). The N-termini of mPERK KD and PKR KD are labeled. Helix αC2 in mPERK KD is also labeled. The disordered regions are represented by dashed lines. (b) Superimposition of mPERK KD C-lobe with active PKR KD C-lobe (left) and inactive PKR KD C-lobe (right). mPERK molecules are colored gold and PKR molecules are colored silver. Phosphorylated Thr residues are shown as ball-and-stick models. The missing parts of the mPERK activation loop are indicated by dashed lines.

Figure 3

Stabilization of the activation loop and helix αG by the phosphorylated Thr980. Ribbon representations of loops and helices are colored pink and cyan, respectively. (a) The activation loop can be stabilized by the charged interactions formed by the phosphate moiety of Thr980 and three basic residues: Lys631, Arg634 and Arg934. All residues involved in the interactions are shown as ball-and-stick models. (b) Helix αG is stabilized by its interactions with the activation loop. All residues involved in the interactions are shown as ball-and-stick models.

Figure 4

mPERK KD may use interdimer interactions to perform autophos­phorylation. The catalytic C-lobes within the PERK homodimer are shown in this figure. One AMP-PNP molecule has been manually modeled into one mPERK protomer and is shown as a ball-and-stick model. The putative catalytic site is indicated by the arrow. A cartoon drawing of the mPERK KD dimer is also shown at the bottom of the figure.

Figure 5

The ‘line-up’ model for PERK pathway activation. (1) Inactive PERK dimers associate with the ER chaperone Bip under nonstressed conditions. (2) Unfolded protein (solid red line) binds the PERK luminal domain dimers through the MHC-like grooves (blue bars) and lines them up. Bip molecules are released from PERK and the activation loops within PERK KD are phosphorylated in a trans-interdimer fashion. The phosphate group is indicated by the solid red circle. (3) Phosphorylation of the activation loop stabilizes both the loop itself and helix αG in the C-lobe. The active PERKs are ready for substrate binding. (4) eIF2α is recruited by the activated PERK and phosphorylated at the Ser51 position by PERK KD.