Crystals of membrane proteins occasionally contain lipid molecules. For example, the structure of bacteriorhodopsin (bR) from lipid cubic phase crystallization revealed 13 phytanyl lipids, seven of which formed a bilayer structure, and a squalene 25
. These, and all other lipids found in crystal structures to date (78 lipids in total 26
), originate from the native membrane, from which they co-purify with the crystallized membrane protein. Such tightly bound lipids have been found to be essential for the structural integrity and activity of a number of membrane proteins 27
None of the AQP 3D crystals examined so far contain lipids, and 2D crystals of AQPs can form with a variety of different lipids, suggesting that AQPs have neither a requirement for specific lipids nor high-affinity lipid binding sites. Nevertheless, our density map revealed that between the AQP0 tetramers are horseshoe-shaped features characteristic of lipid molecules (). Indeed, close inspection revealed that lipids bridge all the contacts between tetramers within a layer and that the tetramers have essentially no direct lateral interaction. In composite omit maps, we could identify nine lipids per AQP0 monomer, which we modeled as complete or partial molecules of dimyristoyl phosphatidyl choline (DMPC, the lipid used for 2D crystallization) (). Phospholipid headgroups have a chiral center at C2 of the glycerol, and the DMPC we used is a racemic mixture. Density is weak or absent at most C2 positions in our map, and often at the attached ester group as well, suggesting that there is little or no selectivity for the biological enantiomer. Very strong density for the phosphate groups, weaker but well defined density for the trimethyl amine groups of the cholines, and unambiguous density for the acyl chains allowed us to build and refine a model in which we chose an enantiomer for each lipid more or less arbitrarily. We have not yet attempted to refine the two alternatives with 50% occupancy each. We have annotated these lipids as PC1 to PC9 (; Suppl. Fig. 6). PC1 to PC7 have extensive protein contacts and appear to represent “annular lipids” immediately adjacent to a membrane embedded protein. PC8 and PC9 are not in contact with protein and thus represent bulk lipids. A detailed description of protein-lipid contacts is provided in Supplementary Materials. As AQP0 has no tight lipid binding sites, interactions between the annular lipids and the AQP0 subunits are likely to represent the kind of contacts that occur between any membrane protein and the lipids surrounding it.
Figure 4 : Lipid-protein interactions in double-layered AQP0 2D crystals. a. Vertical slab through the 2Fo-Fc density map with modelled lipid molecules, revealing the two lipid bilayers in the double-layered AQP0 2D crystal. b. The nine lipids surrounding an AQP0 (more ...)
Annular lipids must adapt to the irregular surface of a transmembrane protein to create a smooth interface for bulk lipids. This fit limits the mobility (and perhaps the chemistry) of annular lipids, as their conformations are partially defined by the protein surface. In our 2D arrays, most of the annular lipids are sandwiched between two tetramers and thus mediate lattice interactions (Suppl. Fig. 7). This packing further restricts their conformations. The cell dimensions of our reconstituted junctions are the same as those in thin junctions between lens fibre cells 28
. We therefore suggest that the lipid-protein interactions we observe in our 2D crystals with the artificial lipid DMPC are representative of those formed by AQP0 tetramers with native lipids in lens fibre cell membranes.
The lipids form a one-molecule wide annular shell around the protein. The positions of the headgroups vary by only ±2 Å in the direction perpendicular to the membrane plane, with a separation of about 34 Å from phosphate to phosphate. The dimensions of the bilayer correspond closely to those of fully hydrated, fluid phase DMPC 29
. A hydrated network of hydrogen bonds and salt bridges holds the lipid phosphates in place. Protein groups interacting with phosphates include three arginine side chains, a tyrosine hydroxyl that mediates one of the arginine contacts, a lysine, a tryptophan indole nitrogen, a glutamine side-chain amide, and at least one main-chain amide. Similar interactions have been described for specifically bound lipids 30
Acyl chains fill the gaps between adjacent tetramers. Their conformations clearly adapt to the knobs and grooves of the apposed hydrophobic protein surfaces. illustrate three examples. PC1 in the extracellular leaflet is the best ordered of the nine DMPC molecules. Its acyl chains are nearly fully extended, packed against those of PC2 and PC3 and sandwiched between five non-polar side chains from one AQP0 and three from the other. PC5 in the cytoplasmic leaflet has somewhat less extended acyl chains. The phosphate receives a hydrogen bond from the indole nitrogen of Trp 10 and Lys238 (as well as the poorly ordered N-terminal segment) of an adjacent subunit. The acyl chains, packed between those of PC4 and PC6, contact four hydrophobic side chains from one subunit (including the hydrophobic face of Trp10) and three from another. PC6, also in the cytoplasmic leaflet, has widely splayed acyl chains, separated by side chains from the two apposed AQP0 molecules. Phe 14 of one molecule and Leu 217 of another are in van der Waals contact through the gap: the only direct interaction between tetramers within a layer.
PC8 and PC9 lie near the fourfold axis. They do not contact protein and thus represent bulk lipids. Neither is as well ordered as the annular lipids. Indeed, PC8 (in the cytoplasmic leaflet) is probably only statistically ordered (two, rather than four, molecules about a fourfold), as there is space for only one of the two acyl chains and no density for the headgroup. The headgroup of PC9 lies about 3 Å closer to the midplane of the bilayer than those of the four other extracellular leaflet lipids; the bilayer thickness may therefore be influenced by adjacency to the protein.