Interactions formed between adjacent AQP0 tetramers and lipid-AQP0 surface complementarity. (A) Equilibrium MD simulations were performed with pairs of AQP0 tetramers without cholesterol (top row) or with cholesterol (bottom row) at the interface. Density maps were calculated for different components next to the AQP0 surface over time and these maps were projected onto the surface of one of the AQP0 tetramers to represent their localization. For clarity, the second tetramer, which would be in front of the shown tetramer, is not shown. The density displayed at arbitrary density units is color-coded and shows the position of cholesterol (red), protein, i.e., the neighboring AQP0 tetramer (brown), sphingomyelin (green) and water (blue). (B) Tij is defined as the fraction of time during which residues (i, j) from opposite tetramers are in contact. This quantity was extracted from the equilibrium simulations for the pure sphingomyelin interface Tij(SM) and for the interface containing cholesterol Tij(Chol). Tij = 0 means that i and j were never in contact and Tij = 1 means that they were always in contact. (C) The pairwise difference ΔTij = Tij(Chol) - Tij(SM) is shown, discarding insignificant changes (ΔTij < 1 percentage, %, points). Accordingly, a value of ΔTij > 0 (ΔTij < 0) corresponds to protein–protein contacts that were more often observed in the simulations with cholesterol (sphingomyelin). For instance, the residue pair Gln129–Ser106 was observed almost 20 percentage points more time in the simulations with cholesterol. The color of the bars indicates the location of the residues (grey: inner part of the extracellular leaflet, i.e., where deep cholesterol resides; white: the rest of the interfacial protein surface, see inset at lower right). Residues involved in a high ΔTij > 10 percentage points are highlighted in orange in the inset (yellow for the contact Leu217–Phe214 observed in the electron crystallographic structure). (D) The average duration for every established protein–protein contact, <τ>ij, is also displayed for the two different lipid interfaces. In (B) and (D), the horizontal dashed lines indicate the highest value observed for all possible residue pairs. Contacts observed in the electron crystallographic structures are highlighted in bold letters. In B–D, the avg±s.e.m. is presented (n = 20, i.e., 10 independent simulation times with 2 symmetric monomeric interfaces). (E) The schematic drawing depicts a top view of the two associated AQP0 tetramers (squares, “Tet 1” and “Tet 2”) with the two central lipids sandwiched between them (black circles). The other lipids at the interface are not shown for clarity. The respective monomer surfaces S1 and S2 are indicated. The region of the surface of the two tetramers that is in total covered by a lipid, AContact (red line) was normalized by the surface area of the lipid, ALipid (here corresponding to the perimeter of the circles). This ratio gives a measure of the surface complementarity between the tetramers and the sandwiched lipids, i.e., the higher the value of AContact/ALipid the more the two surfaces complement each other. (F) Normalized histograms of AContact/ALipid obtained from the equilibrium MD simulations are shown for the central sphingomyelin (SM) and cholesterol (Chol). See also Figure 8–figure supplements 1–3.