Otoferlin C2F Domain-Induced Changes in Membrane Structure Observed by Sum Frequency Generation

Abstract

Proteins that contain C2 domains are involved in a variety of biological processes, including encoding of sound, cell signaling, and cell membrane repair. Of particular importance is the interface activity of the C-terminal C2F domain of otoferlin due to the pathological mutations known to significantly disrupt the protein's lipid membrane interface binding activity, resulting in hearing loss. Therefore, there is a critical need to define the geometry and positions of functionally important sites and structures at the otoferlin-lipid membrane interface. Here, we describe the first in situ probe of the protein orientation of otoferlin's C2F domain interacting with a cell membrane surface. To identify this protein's orientation at the lipid interface, we applied sum frequency generation (SFG) vibrational spectroscopy and coupled it with simulated SFG spectra to observe and quantify the otoferlin C2F domain interacting with model lipid membranes. A model cell membrane was built with equal amounts of phosphatidylserine and phosphatidylcholine. SFG measurements of the lipids that make up the model membrane indicate a 62% increase in amplitude from the SFG signal near 2075 cm^-1 upon protein interaction, suggesting domain-induced changes in the orientation of the lipids and possible membrane curvature. This increase is related to lipid ordering caused by the docking interaction of the otoferlin C2F domain. SFG spectra taken from the amide-I region contain features near 1630 and 1670 cm^-1 related to the C2F domains beta-sandwich secondary structure, thus indicating that the domain binds in a specific orientation. By mapping the simulated SFG spectra to the experimentally collected SFG spectra, we found the C2F domain of otoferlin orients 22° normal to the lipid surface. This information allows us to map what portion of the domain directly interacts with the lipid membrane.

Figure 1

(A) Representative image of a co-floatation assay with the C2F domain of otoferlin mixed with DPPS/DPPC (1:3) or DPPC SUV in the presence of calcium or EDTA. (B) Quantification of the results of the liposome binding assay is shown. S denotes supernatant, and B denotes protein bound to floated liposomes. To see this figure in color, go online.

Figure 2

Depicts CD region ssp polarization combination SFG spectrum of (A) the 1:1 molar ratio d62-DPPS and d62-DPPC lipid monolayer before (black) and after (red) otoferlin C2F domain is injected into the subphase, and (B) a cartoon schematic of the otoferlin C2F domain interacting with the lipid monolayer is shown. The schematic depicts the lipid dimpling after the C2F domain of otoferlin adsorbs to the lipid monolayer. The pure d62-DPPC lipid monolayer (C) before (black) and after (red) otoferlin C2F domain is injected into the subphase, and (D) a cartoon schematic of the otoferlin C2F domain interacting with the lipid monolayer is shown. Spectra are offset for clarity, and the blue line represents the baseline. To see this figure in color, go online.

Figure 3

Structure of the otoferlin C2F domain from PDB 4IQH (A). Shown are the coordinate system and Euler angles of the protein orientation in the laboratory frame (B). Shown is the experimental setup for in situ SFG measurements of OtoF-bilayer binding (C). To see this figure in color, go online.

Figure 4

(A) Optimal fit of the experimental ppp (black) and ssp (red) data of OtoF, for a protein tilt angle θ = 22 ± 2° and a twist angle ψ = 74 ± 2°. (B) Shown is an error-weighted two-dimensional residual sum-of-squares (2D-RSS) plot of the amide-I (1600–1700 cm⁻¹) region composed of ∼10,000 spectral calculations at a θ and ψ resolution of 2.5° that indicates that the experimental spectra can only be modeled well for a small range of protein orientations. In the white area, the error-weighted RSS is more than twice the minimal error-weighted RSS value (255). The 2D-RSS plot for the whole modeled spectral range (1440–1740 cm⁻¹) can be found in Fig. S4. To see this figure in color, go online.

Figure 5

Extracted orientation from the raster search. (A) Average orientation for OtoF on a lipid bilayer surface from SFG spectra calculations show significant spectral match with experimental spectra. (B) Zoomed in view of the otoferlin C2F domain binding loops with the aspartic acids is shown. To see this figure in color, go online.