Comparison of our new AQP0EPL
structure with the previously determined AQP0DMPC
structure shows that the annular lipids studied here have very little influence on the structure of AQP0. With only a few exceptions, such as Phe 14, the amino-acid residues in the AQP0EPL
structures, in particular the ones lining the water channel, have nearly identical conformations. The virtually identical channel structure in AQP0EPL
is consistent with a previous study that showed that the lipid environment did not affect water conduction by AQP1 (Zeidel et al, 1994
), although only a very limited range of lipid compositions were tested in this study.
Electron crystallography of AQP0 2D crystals makes it possible to visualize the lipids surrounding the membrane protein. In our studies, only a single layer of lipid molecules separates the individual tetramers in the 2D crystals (). This tight packing of the lipids in the 2D crystals restricts their mobility and makes it possible to see the lipids in the density maps, providing us with the opportunity to study the interactions of annular lipids with AQP0. However, this situation is not typical for a biological membrane, in which membrane proteins tend to be separated by a larger number of lipids. Nevertheless, the 2D crystals formed in vitro
display the same lattice parameters as the 2D arrays formed by AQP0 in native lens membranes (Buzhynskyy et al, 2007
). The 2D crystals are therefore a good representation of the AQP0 arrays in the lens membrane. Furthermore, AQP0 forms 2D crystals with lipids as different as DMPC and EPLs. The sandwiching between two adjacent tetramers may thus limit the mobility of the lipids but may not impose further restrictions on the behaviour of the lipids surrounding AQP0. Our findings concerning the interaction of annular lipids with AQP0 in the 2D crystals should therefore bear relevance for membrane proteins surrounded by a larger number of lipids. Nevertheless, further studies will be required to confirm this notion.
The structures of AQP0 in two different lipid environments allow us to see how the protein and the lipid bilayer adapt to each other. To accommodate proteins in a lipid bilayer with a different hydrophobic thickness, a situation known as hydrophobic mismatch, it has been proposed that either the lipids adjust their length to match the protein or that α-helical membrane proteins may stretch or contract axially to adapt to the thickness of the surrounding lipid bilayer (Lee, 2004
). It has been shown for several membrane proteins that hydrophobic mismatch affects their activity (Lee, 2004
), suggesting changes in protein structure. It is not clear, however, whether all membrane proteins have the flexibility to adjust to the hydrophobic thickness of the surrounding lipid bilayer. Aquaporin-0, which is a very stable and presumably a very rigid membrane protein, has the same structure in the EPL and DMPC bilayers (). In the two structures, it is exclusively the lipids that adapt to the protein, which bend and interdigitate to accommodate the longer acyl chains of the EPLs. A caveat of this observation is that the acyl chains of EPLs are not only longer than those of DMPC, but 55% of the acyl chains of EPLs are unsaturated (Lugtenberg and Peters, 1976
), and double bonds reduce the hydrophobic thickness of the bilayer formed by unsaturated lipids (Marsh, 2008
). Thus, although the AQP0DMPC
structures suggest that it is exclusively the lipids that adapt to the protein, the hydrophobic thickness of the DMPC and EPL bilayers may be too similar to induce observable changes in the AQP0 structure. To conclusively rule out structural changes in AQP0 due to hydrophobic mismatch, it will be necessary to determine structures of AQP0, in which the protein is surrounded by lipids that form bilayers with a hydrophobic thickness that is significantly different from that of AQP0.
After modelling all the headgroups as ethanolamine and refining the lipids in the AQP0EPL
structure, we did not observe any density that indicated PG or CL headgroups. This result suggests that there are no lipid positions on the surface of AQP0 that are preferentially occupied by PG or CL lipids. In the absence of preferential binding sites, averaging of the three headgroups would probably result in all headgroups appearing as the predominant PE headgroup. Furthermore, inspection of the protein surface did not reveal specific CL-binding sites as defined by Palsdottir and Hunte (2004)
. As CL, a lipid composed of four acyl chains and two phosphodiester groups linked by a glycerol, constitutes only 10% of EPLs, it may randomly occupy two adjacent lipid positions and thus be obscured as a result of averaging. Alternatively, due to its larger size, CL may preferentially localize to the larger lipid area in the middle of four neighbouring AQP0 tetramers in which no ordered lipid molecules were found (asterisks in ). As PG and CL lipids do not occur in lens membranes, it is not surprising that AQP0 does not select for these lipids at particular positions.
Despite the different acyl chain length and the different orientations of the headgroups, the average distance between the phosphodiester groups in the two leaflets is almost identical in the bilayers formed by EPLs, 31.9 Å, and DMPC, 33.6 Å (). More surprisingly, the average distance between the C2 atoms of the glycerol of the lipids in the two leaflets is larger for the shorter DMPC lipids, 31.2 Å, than for the longer EPLs, 27.0 Å (). This finding suggests that the vertical position of the lipids is defined by the strong charges of the phosphodiester groups that have to be positioned outside the hydrophobic belt of the membrane protein. The glycerol backbone, however, which is largely hydrophobic in nature, is allowed more flexibility in its positioning within the hydrophobic region of the membrane protein and can occupy the same region as the acyl chains.
In conclusion, we propose that the vertical position of the annular lipids surrounding a membrane protein is defined by the thickness of the hydrophobic surface of the membrane protein, with the charged phosphodiester groups of the lipids in the two leaflets being positioned immediately outside of the hydrophobic belt of the protein. Although some membrane proteins may change their structure to adjust to bilayers with a significantly different hydrophobic thickness, it remains to be determined whether rigid membrane proteins, such as AQP0, can adapt in a similar manner. The requirement that acyl chains fill in the gaps on the protein surface constitutes the guiding principle for the lateral interactions of annular lipids with membrane proteins. Additional interactions of the protein with headgroups of annular lipids, that is, lipid-binding sites, may allow membrane proteins to select for specific lipids and/or to form tight interactions with lipids important for their function or structural integrity. Furthermore, the fact that AQP0 forms well-ordered 2D crystals with lipids as different as DMPC and EPLs suggests that it will be possible to grow 2D crystals of AQP0 with many other lipids, opening an avenue for the systematic study of lipid-protein interactions.