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in between two AQP0 tetramers, which is not typically the

case for lipids surrounding membrane proteins in biological

membranes.

The tight positioning of the lipids in AQP0 crystals reduced

their mobility and rendered them visible in the density maps,

making it possible to describe the lipidprotein interactions.

If AQP0 2D crystals could be grown with other lipids, such

crystals would provide an opportunity to study how a mem-

brane protein interacts with different lipids in a near-native

environment. While formation of high-quality 2D crystals

usually requires the use of a specific lipid, aquaporins can

often form 2D crystals with a variety of lipids. AQP1, for

example, formed large, well-ordered 2D crystals with three

different lipids (Jap and Li, 1995; Murata et al, 2000; Ren

et al, 2000). In the case of AQP0, 2D crystallization screens

showed that, in addition to DMPC, AQP0 also forms 2D

crystals with E. coli polar lipids (EPLs). E. coli polar lipids

differ from DMPC in headgroup chemistry, as well as acyl

chain length and saturation. The headgroups of EPLs are a

mixture of phosphatidylethanolamine (PE), phosphatidylgly-

cerol (PG) and cardiolipin (CL) rather than the pure phos-

phatidylcholine (PC) headgroup of DMPC. The acyl chains of

DMPC are two saturated 14-carbon acyl chains, whereas the

average length of the acyl chains of EPLs is 16 carbon atoms,

and approximately 55% of the acyl chains are unsaturated

(Lugtenberg and Peters, 1976). We have now optimized the

quality of the AQP0 2D crystals formed with EPLs and

produced a density map at 2.5 A

° resolution. As with the

previous density map produced with 2D crystals formed

with DMPC, the current density map revealed the seven

annular lipids, allowing us to compare the interactions of

AQP0 with two very different lipids, DMPC and EPLs.

Results

Structure of AQP0 in DMPC and EPL bilayers

Well-ordered, double-layered 2D crystals of AQP0 formed

with EPLs at a lipid-to-protein ratio of 0.25 (mg/mg;

Figure 1A). These crystals were used to record electron

diffraction patterns at liquid helium temperature (

B6 K).

Diffraction patterns from untilted crystals showed reflections

beyond 1.9 A

° (Supplementary Figure S1), but the resolution

was more limited in diffraction patterns of highly tilted

crystals (Supplementary Figure S2). After merging 281 dif-

fraction patterns and phasing by molecular replacement, we

produced a density map at 2.5 A

° resolution (Figure 1B) and

modelled and refined the structure of AQP0 (residues 7226)

in the membrane junction (Figure 1C; Table I). Residues 16

and 227263, which include the C-terminal helix modelled in

the previous structure of AQP0 in a DMPC bilayer (Gonen

et al, 2005), did not show clear density in the map and were

therefore excluded from the model of AQP0 in the EPL

bilayer.

Other than the C-terminal helix, which was disordered and

therefore could not be modelled, the structure of AQP0 in the

EPL bilayer (AQP0EPL) is virtually identical with its structure

in the DMPC bilayer (AQP0DMPC; Figure 1D). The r.m.s.d.

values between all modelled backbone atoms is 0.48 A

° ,

and 0.4 A

° if only the transmembrane domains are compared.

The pore-lining residues in AQP0EPL and AQP0DMPC are also

essentially the same, and both structures show three water

molecules in the centre of the channel at the same positions

(Figure 1E).

Modelling the EPL bilayer

In addition to the protein, the density map also allowed us to

build all seven annular lipids surrounding each AQP0 mono-

mer, but not the lipids in the central area between four

adjacent tetramers (asterisks in Figure 2A). The acyl chains

of the annular lipids were initially modelled as 10 carbon

chains and then extended to occupy the maximal length that

was clearly resolved in the density map. In the initial density

map, densities representing the acyl chains were often

branched, indicating that they can adopt multiple conforma-

tions. As the data were insufficient for the refinement of

alternative conformations, we chose to build each acyl chain

into the strongest density, representing the predominant

lipid conformation. After several rounds of refinement, the

Figure 1 Double-layered 2D crystals of AQP0 in Escherichia coli polar lipids (EPLs). (A) Representative AQP0 2D crystal formed with EPLs in

negative stain. Scale bar: 1 mm. (B) Region of the final 2FoFc map refined to 2.5 A

° resolution showing pore-lining residues and a water

molecule. (C) Atomic model of the AQP0 membrane junction. (D) Overlay of the AQP0EPL (gold) and AQP0DMPC (light blue) structures. (E)The

water pore in AQP0EPL (gold) and AQP0DMPC (light blue). The three water molecules in the pore of AQP0EPL (red) are at similar positions as those

previously seen in AQP0DMPC (blue). The 2FoFc density map for the water molecules is shown at a contouring level of 1s (blue wire mesh).

Interaction of AQPO with E. coli lipids

RK Hite et al

2010 European Molecular Biology Organization

The EMBO Journal

VOL 29 | NO 10 | 2010 1653