Exploring the structural dynamics of Ire1’s AH during activation by lipid bilayer

In order to gain insights in to the structural dynamics of the AH (Ire1^534-539) in different membrane environments, a spin-probe was installed at six consecutive positions in the minimal sensor construct. After reconstituting this construct in two distinct membrane environments corresponding to lipid composition 1 and 8, low temperature cwEPR spectra (-115°C) were recorded to gain information on the average inter-spin proximity and the polarity of the spin probe’s environment. cwEPR spectra of the spin labeled mutants Y536C to I539C

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were broadened in lipid composition 8 as compared to lipid composition 1 (Fig. 45 A and B), thereby corroborating findings with the minimal sensor labeled at the position of R537 or at the endogenous C552 (Fig. 44). Again, these data suggest that the minimal sensor consisting containing the AH and the TMH of Ire1 shows a membrane-sensitive oligomerization, which is likely to support the oligomerization of Ire1 under conditions of lipid bilayer stress. No spectral broadening, however, was observed when the spin probe was introduced more N-terminally at the positions L534 and V535 (Fig. 45 A and B). This finding suggests an inter-spin distance greater than 2.0 nm at these positions even in oligomers of Ire1.

Analysis of the low temperature cwEPR spectra (-115°C) can also provide information on the polarity of the spin probe’s nano-environment based on the hyperfine splitting (Azz). In order to derive values for the hyperfine splitting Azz, the magnetic field difference between the low and high field peaks can be determined, to yield the polarity value 2Azz (Bordignon and Steinhoff, 2007). In order to test how individual residues of the AH interact with the lipid matrix and in order to test for major structural changes in the AH region when the minimal sensor is reconstituted in different membrane environments, the 2Azz value was extracted for the spin probes installed at the residues Y536 to I539 with the minimal sensor reconstituted in lipid composition 1 and 8. The higher the 2Azz value, the more polar the nano-environment of the spin probe (Bordignon and Steinhoff, 2007).

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Figure 45 | cwEPR spectra of MBP-Ire1^AH+TMH labeled at the indicated residues and reconstituted in distinct membrane environments.

(A) A representative, intensity normalized cwEPR spectrum recorded at -115°C illustrates how the 2Azz value is derived from the spectra. (B) The minimal sensor was labeled at the indicated resdues and reconstituted in liposomes of different lipid environments illustrated by the previously introduced color code. cwEPR spectra were recorded at -115°C and plotted after intensity normalization. (C) The semi-quantitative index Lf/Mf was derived from cwEPR spectra shown in (B) and plotted against the position of labeling. Higher Lf/Mf values indicate low average inter-spin distances. The error bars represent the average ± SEM for 3 independent experiments. Significance was tested by an unpaired student’s t-test. ***p<0.001, **p<0.01, *p<0.05. (D) The 2Azz tensor is derived from cwEPR spectra and plotted against the position of labeling. The error bars represent the average

± SEM for 3 independent experiments. Significance was tested by an unpaired student’s t-test. **p<0.01.

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When the polarity value 2Azz was plotted against the position of labeling, a helical pattern of polarity was observed in both lipid environments (Fig. 45 C). This validates that the predicted AH indeed forms a helix with a more polar and apolar environment. Moreover, this shows that the structure of the AH does not change in rather distinct lipid environments. While the AH is crucial to drive the minimal sensor into oligomers (Fig. 41 - 45), it does not undergo a major change of its secondary structure. The nano-environment of the spin probe was less polar for residues located in the hydrophobic face (V535, I538, I539) than for those pointing towards the hydrophilic face of the AH (L534, R537). The overall polarity of the spin probes did not change remarkably in response to changes in molecular lipid packing. Only for the reporter residue at position R537 the polarity increased in densely packed liposomes (Fig. 45 C). As the average distances of the R537 reporter changed remarkably in different lipid environments, this residue might be at the interface of two Ire1 molecules within the dimer, which naturally would affect the polarity at this position.

Taken together, these data suggest that the AH of Ire1 is stably inserted into the lipid bilayer, irrespective of the lipid packing density. This implicates, that the overall architecture of Ire1’s AH overlapping with and adjacent to the TMH is not perturbed by the lipid environment yet sufficient to drive Ire1 in to oligomers during lipid bilayer stress.

Figure 46 | Representative structures of the initial and final configuration of the Ire1 minimal sensor in MD simulations.

Structures from MD simulations of the wild type minimal sensor (Ire1 minimal sensor, orange; POPC and DOPC, grey, cholesterol, green). Representative structures of the wild type minimal sensor modeled as a straight helix into the lipid compositions 1 and 7 from in vitro experiments. MD simulations were performed and analyzed by Roberto Covino & Gerhard Hummer, Institute for Theoretical Biophysics, MPI for Biophysics.

Atomistic molecular dynamics (MD) simulations of the minimal sensor of Ire1 (Ire1^526-561) were performed in lipid bilayers corresponding to lipid compositions 1 and 7 of the in vitro reconstitution experiments (performed by Roberto Covino & Gerhard Hummer, MPI for Biophysics). In these simulations, the hydrophobic portion of the AH entered the lipid bilayer and remained stably inserted throughout the > 3 µs simulation with the hydrophilic portion of the AH facing the aqueous environment (Fig. 46). Intriguingly, the membrane integration of

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the AH forced the TMH of Ire1 into a strongly tilted orientation relative to the lipid bilayer and introduced a temporary kink in the minimal sensor.