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. 2015 Jun 3;34(11):1589-600.
doi: 10.15252/embj.201489183. Epub 2015 Apr 28.

Crystal structures reveal transient PERK luminal domain tetramerization in endoplasmic reticulum stress signaling

Marta Carrara  1 , Filippo Prischi  1 , Piotr R Nowak  1 , Maruf Mu Ali  2
Affiliations

Crystal structures reveal transient PERK luminal domain tetramerization in endoplasmic reticulum stress signaling

Marta Carrara et al. EMBO J. .

Abstract

Stress caused by accumulation of misfolded proteins within the endoplasmic reticulum (ER) elicits a cellular unfolded protein response (UPR) aimed at maintaining protein-folding capacity. PERK, a key upstream component, recognizes ER stress via its luminal sensor/transducer domain, but the molecular events that lead to UPR activation remain unclear. Here, we describe the crystal structures of mammalian PERK luminal domains captured in dimeric state as well as in a novel tetrameric state. Small angle X-ray scattering analysis (SAXS) supports the existence of both crystal structures also in solution. The salient feature of the tetramer interface, a helix swapped between dimers, implies transient association. Moreover, interface mutations that disrupt tetramer formation in vitro reduce phosphorylation of PERK and its target eIF2α in cells. These results suggest that transient conversion from dimeric to tetrameric state may be a key regulatory step in UPR activation.

Keywords: ER stress; PERK; cell signaling; unfolded protein response.

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Figures

Figure 1
Figure 1
Human PERK luminal domain tetramer structure

  1. Cartoon representation of human PERK LD tetramer viewed from top along twofold rotation axis with monomer A in orange, monomer B in magenta, monomer C in cyan, and monomer D in green.

  2. Top view of human PERK tetramer in molecular surface representation.

  3. Side view of tetramer displaying the tetramer interface.

  4. Front view of PERK tetramer showing the dimerization interface between monomers. The dimerization interface is offset from the twofold rotation axis by ˜25°.

  5. Dimer component of PERK tetramer illustrating the concave surface as viewed from top.

  6. Cartoon representation of an individual PERK LD monomer divided into subdomains with red representing dimerization subdomain, blue the β-sandwich subdomain, and yellow tetramer subdomain. Each secondary structural element is numbered.

Figure 2
Figure 2
The novel tetramer interface

  1. Residues from PERK monomer that are involved in the tetramer interface. The residues lining the binding cleft and helix α2 are predominantly hydrophobic.

  2. Overview of molecular interactions between monomers, colored in cyan and orange, that are involved in tetramer interface.

  3. View along the helix α2 shows the hydrophobic core interaction between the swapped helix and the binding cleft.

  4. Alignment of PERK and Ire1 sequences from both human and mouse species. The red-colored letters denote functionally conserved residues, with green stars indicating residues involved in hydrophobic core interactions, while blue triangles indicate residues that contribute to the tetramer interface via hydrogen bond interactions.

  5. Electrostatic surface potential representation of PERK monomer showing tetramer interface.

Figure 3
Figure 3
Mouse PERK luminal domain dimer structure

  1. Transparent molecular surface representation of dimeric mouse PERK LD crystal structure, with monomer A colored in red and monomer B in yellow.

  2. Mouse PERK monomer organized into three subdomains with dimerization domain in red and β-sandwich domain in blue. The tetramer subdomain is disordered and is not visible within the structure.

  3. Structural superimposition of mouse PERK (red) and human PERK (cyan) monomers. The rmsd value between the two monomers is 1.1 Å.

  4. Structural superimposition of mouse PERK (red) and human PERK (cyan) dimers, with an rmsd value of 1.0 Å, suggests that the alignment of the dimer interface is consistent between PERK species.

Figure 4
Figure 4
Comparison of PERK and Ire1 luminal domains structures

  1. Secondary structure comparison of dimerization subdomain interface between PERK and Ire1. PERK dimer interface is greater in area compared to Ire1 due to better alignment of monomers.

  2. Structural superimposition of human PERK (cyan) with human Ire1 (red) crystal structures. The α2 helix in PERK structure is projected outwards to form the helix-swapped tetramer interface. The equivalent helix in Ire1 is shorter and projected downward; this orientation is not conducive for helix swap to occur between monomers. The distinctively long helix (αB) observed in Ire1 structure is not present within the PERK structures—a point that is further supported by sequence alignments showing PERK lacking the long helix (αB) region (Supplementary Fig S2), and is not involved in tetramer formation.

  3. A section from a structural pairwise alignment between human PERK and yeast Ire1 crystal structures (Supplementary Fig S3) reveals that the only significant stretch of identity (NSVYL-motif) occurs on β18, which forms the base of the cleft within the tetramer subdomain.

Figure 5
Figure 5
Small angle X-ray scattering (SAXS) analysis of PERK luminal domain in solution

  1. Sedimentation velocity AUC profile reveals human PERK LD exists as a dimer–tetramer species in solution, in a dimer3:2tetramer ratio, indicating the transient nature of the tetramer species, while reinforcing the stable nature of the dimer interface.

  2. AUC analysis of mouse PERK LD also indicates that mouse PERK LD forms dimer–tetramer species in a similar ratio (dimer3:2tetramer) to that of human PERK LD.

  3. Small angle X-ray scattering analysis of human PERK LD in solution comparing the experimental SAXS profile (gray dots) to the computed profile of PERK LD crystal structures, when using a dimer3:2tetramer ratio of dimer–tetramer, based on AUC and further reinforced by the program OLIGOMER, resulting in an excellent fit (χ = 1.1). Inset, profiles for independent SAXS runs at various concentrations.

Figure 6
Figure 6
Structure-guided mutational analysis in vitro and in vivo

  1. Sedimentation velocity AUC analysis comparing the levels of dimer and tetramer in solution between wild-type PERK luminal domain (black) and tetramer interface mutants: W165A (red), L388N (blue), L395N (green), L397N (cyan), and A378N (magenta). All mutations reduce tetramer formation in vitro, with mutations situated at the base of the hydrophobic cleft (L388N, L395N, L397N) and on the helix α2 (A378N) having the greatest effect.

  2. PERK−/− MEF cells were transfected with empty vector (EV), myc-tagged wild-type PERK (WT), and myc-tagged PERK tetramer mutants, and were assessed for PERK and eIF2α phosphorylation both in the absence and presence of 5 μm tunicamycin for 4 h to induce ER stress. After immunoblotting, we observed a reduction in the levels of PERK and eIF2α phosphorylation for mutants when compared to wild-type PERK that mirrors the effects seen in vitro.

  3. Model illustrating the transition from dimer to tetramer being a likely regulatory step in UPR signal activation. Tetramer formation results in a higher efficiency of auto-phosphorylation of the PERK kinase domain.

Source data are available online for this figure.
See this image and copyright information in PMC

References

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