Figure 7. Equilibrium molecular dynamics (MD) simulations of pairs of associated aquaporin-0 (AQP0) tetramers and force-induced separation of two associated AQP0 tetramers.

(A) Distance between the centers of mass d_CM (top) and minimum distance d_tet-tet (bottom) between the pair of AQP0 tetramers during the 2×AQP0 simulations in equilibrium for the interface containing only sphingomyelin (SM) molecules (SM, green) and the interface containing the deep cholesterol (Chol, blue) (n=10 simulations for each case). (B) Principal component (PC) analysis of the relative movements between the two tetramers. Here, the motion of one of the tetramers (dashed-line rectangle) relative to the other tetramer (solid-line rectangle) was monitored. The three main PC accounted for 67.9% of the total relative motion between tetramers (PC1: 34.3%, PC2: 24.7%, and PC3: 8.9%). The schematic drawings illustrate the three main modes of motion: bending (depicted in the drawing as viewed from the side of the membrane), lateral rotation (depicted in the drawing as viewed from the top of the membrane), and torsion (depicted in the drawing as viewed from the side of the membrane with one tetramer in front of the other). Lipids at the interface between the two tetramers (first two panels) and lipids surrounding the two tetramers (last panel) are shown. PC1 plus PC2 capture the bending and rotation while PC3 corresponds to torsion. The histograms in the bottom panels show the projections of the MD trajectories onto the three main PC vectors, for the interfaces containing only sphingomyelin (SM, green) or containing the deep cholesterols (Chol, blue). The approximate angular extent for each of the modes, attributed to these projections, is indicated (in degrees). The distributions with and without cholesterol are very similar, except for PC1. Nevertheless, PC1 relates to a small angular variation (~7.1° bending together with ~6.5° rotation). (C) Two AQP0 tetramers (white surface) arranged as in two-dimensional (2D) crystals and embedded in a pure SM membrane were pulled apart by exerting a harmonic force F on them in the direction that connects their centers of mass (d_COM). Two different interfaces were studied: the ‘SM interface’ consisted solely of SM lipids (green spheres) as seen in the AQP0_2SM:1Chol structure, whereas the ‘Chol interface’ contained the deep cholesterols seen in the AQP0_1SM:2Chol structure. The reference position of the harmonic springs used to exert the force F was moved at a constant velocity V_pull/2. (D) Force (F) and distance (d_COM) time traces are shown for one of the simulations using a V_pull of 0.004 m/s. A Gaussian smoothing function (black continuous lines) was applied to the curves (yellow and purple). The detachment force (black circle) was computed as the highest recorded force when d_COM started to increase and below a cut-off distance d_COM of 7.3 nm (horizontal dashed line indicates the cut-off distance and the vertical dashed line indicated the time when this value was surpassed). Note that using different cut-off distances did not change the overall trend (see Figure 7—figure supplement 1). The inset shows an example of the arrangement of the tetramers at the moment of detachment. The red circle indicates the last contact. (E) Force–distance profiles for the two different interfaces are presented for the three indicated pulling velocities (n=20 for each case). Dots indicate the point of detachment. (F) Detachment force is presented as a function of the pulling rate for the two interfaces (box plots, n=20). A fit of the form F_detach = A + B*log(V_pull) is shown to guide the eye with lines (F_detach = [2164+232*log(V_pull)] for the ‘Chol’ interface and F_detach = [2116+239*log(V_pull)] for the ‘SM’ interface). p-Values comparing the two datasets, separately for each pulling velocity, are 0.022 (V_pull = 0.004 m/s), 0.015 (V_pull = 0.02 m/s), and 0.262 (V_pull = 0.1 m/s) (Mann–Whitney U test). Furthermore, a two-way ANOVA test, considering the three pulling velocities at once, retrieved a p-value of 0.003 for the lipid interface change (i.e. Chol vs. SM). See also Figure 7—figure supplement 1.

Figure 7—figure supplement 1. Detachment force versus pulling rate for different d_cut-off criteria.

The cut-off value chosen to define tetramer separation does not alter the conclusion that the presence of deep cholesterol at the interface (blue) results in a more stable association between the tetramers (i.e. requiring a high detachment force) than when the interface only contains sphingomyelin (green) (n=20 for each case).