The structural and functional role of Ire1’s TMH in ER stress

To investigate the dimerization interface of Ire1’s TMH within the lipid bilayer, a cysteine cross-linking approach was designed. A cysteine scan along the entire TMH was performed to study the crosslinking efficiency of the minimal sensor MBP-Ire1^AH+TMH reconstituted in a membrane environment with tightly packed lipids. Thus, single cysteine mutants of the minimal sensor were reconstituted in liposomes consisting of EPL and DPPC (7:3), mimicking a densely packed membrane environment.

The quality of the purified protein was examined as previously described (Fig. 36; 5.7.) and reconstituted into EPL:DPPC liposomes. The protein was successfully integrated and proteoliposomes were harvested by centrifugation (Fig. 49 A). Extensive quality control of proteoliposomes was performed to validate proper insertion (Fig. 49 B to D). Sucrose gradients, which were fractionated and analyzed for their protein and lipid contents, revealed that no protein aggregates were present after reconstitution (Fig. 49 B and C). Membrane extraction experiments using Na2CO3 did not extract MBP-Ire1^AH+TMH from the membrane, indicating that the protein was efficiently inserted into the ER-membrane (Fig. 49 D).

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Figure 49 | Reconstitution and quality control of MBP-Ire1^AH+TMH containing proteoliposomes.

Proteoliposomes containing the indicated mutants and indicated lipid mixtures at a molar protein to lipid ratio of 1:6000. (A) Reconstitution of MBP-Ire1^AH+TMH was performed using EPL:DPPC (7:3) liposomes. After reconstitution, the resulting proteoliposomes were pelleted (450.000x g, 90 min) and resuspended in reconstitution buffer. To test the reconstitution efficiency, samples of supernatant (S) and pellet (P) after the reconstitution were taken and subjected to an SDS-PAGE followed by immunoblotting using anti-MBP antibodies. (B, C) The quality of the proteoliposomal preparation was validated by a sucrose step gradient centrifugation (B), using the depicted sucrose step gradient (C) followed by an analysis of the lipid and protein distribution in fractions taken after equilibrium centrifugation. Lipid distribution throughout the gradient was monitored by fluorescence spectroscopic measurements utilizing the Hoechst33342 dye. Protein content was monitored by subjection of samples to an SDS-PAGE followed by immunoblotting using an MBP antibody. (D) Proteoliposome containing samples were treated with 0.1 M Na2CO3 pH 11.0 for extraction of peripherally attached protein from proteoliposomes or 1% Triton X-100 (T-X100) as a solubilization control. Samples of supernatant (S) and pellet (P) after the treatment with different additives were taken and subjected to an SDS-PAGE followed by immunoblotting using anti-MBP antibodies. (B, C, D) Quality control of the minimal sensor in EPL:DPPC liposomes was established and performed in collaboration with Julian Wagner during his Diploma Thesis under the supervision of Kristina Halbleib.

After reconstitution, crosslinking was performed for 6.5 days by leaving the sample in a normal oxygenated atmosphere and without any added crosslinking reagents. Large differences in cross-linking efficiencies were observed depending on the position of the cysteine ranging from ≈ 5 % to 33 % (Fig. 50 A to C). When a cysteine residue was inserted at positions G542, L546, L547, I550 and F551 only 10 % of crosslinking was observed – thus the crosslinking efficiency was low. Cysteine residues at positions E540, V543, L545, F548 and L549 resulted in 15 – 29 % of crosslinking and were classified as residues with intermediate crosslinking efficiency. Finally, cysteine residues at position T541, F544 and C552 resulted in crosslinking is 30% or higher, these residues were classified as residues with high crosslinking efficiency.

Moreover, the overall crosslinking efficiency for the N-terminal part of the TMH (E540 – L545) is higher than that of the C-terminal part of the TMH (L546 – C552) (Fig. 50 C). This finding indicates that the AA residues in the N-terminal part of the TMH are in closer proximity to each other than the residues in the C-terminal part of the TMH.

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Figure 50 | In vitro crosslinking studies of the reconstituted minimal sensor.

The predicted TMH (black) and juxtra-membrane regions (grey) of Ire1. Residues with a crosslinking efficiency below 10% are depicted in black, those over 30% are highlighted in red, intermediate crosslinking residues in salmon (B) Helical wheel analysis (Wenxiang Diagram; (Chou, 2011) of the TMH (Ire1^538-555) of Ire1. Residues are color coded as introduced in (A), the N- and C-terminal residue of respective projections are labeled. (C) Proteoliposome containing samples were incubated in an oxygen containing environment for 6.5 days at 37°C. Dimerization was monitored by subjection of samples to an SDS-PAGE followed by immunoblotting using anti-MBP antibodies. The dimer content was quantified in respective samples. Residues at positions showing crosslinking efficiencies of 30% or higher (red), intermediate crosslinking efficiency 15%<X<30% (salmon) and <15%

(grey) are depicted. Crosslinking experiments were established in collaboration with Julian Wagner during his Diploma Thesis under the supervision of Kristina Halbleib. Data sets of both experimenters were merged. The error bars represent the average

± SEM for 3 independent experiments.

This crosslinking approach revealed that the TMHs do not just face each other in random orientations. Some residues do not crosslink and therefore do not seem to come in close contact. These data may be interpreted structurally and could be used as constraints for building a model for dimeric TMHs. A particularly tempting model would be the formation of X-shaped dimer, in which the tilted TMHs cross each other in the core of the bilayer around the position of F544C, which showed particularly strong crosslinking. Interestingly, such a conformation has been observed in preliminary MD simulations (Fig. 51) performed by Roberto Covino & Gerhard Hummer from the MPI for Biophysics in Frankfurt.

100

L545C L546C L547C F548C L549C I550C F551C C552

C552S E540C T541C G542C V543C F544C L545C L546C L547C F548C L549C I550C F551C C552

0

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Figure 51 | MD simulations reveal the formation of a X-shaped dimer.

Representative structure of the Ire1-derived minimal sensor, represented as in Fig. 46. in the densely packed lipid environment 7. MD simulations were performed by Roberto Covino & Gerhard Hummer, Institute for Theoretical Biophysics, MPI for Biophysics.

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