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Interaction of the lipid acyl chains with AQP0

In contrast to the headgroups, acyl chains seem to be the

crucial element guiding the interaction of annular lipids with

the protein. Comparison of the lipids in AQP0EPL and

AQP0DMPC reveals that the acyl chains in the extracellular

leaflet occupy quite similar positions. The acyl chains of PE3

and PE4 extend along the same paths as those of the

corresponding lipids, PC3, and PC4. The two acyl chains of

PE2 are in similar positions as those occupied by one of the

acyl chains of PC1 and one of the acyl chains of PC2

(Figure 3C and Supplementary Video S1).

The positions of the acyl chains in the cytoplasmic leaflet

vary to a greater degree between AQP0EPL and AQP0DMPC

than those of the acyl chains in the extracellular leaflet

(Figure 3C). The gap between two adjacent AQP0 tetramers

is narrower on the extracellular side of the bilayer, which may

force the lipids in this leaflet into more defined positions than

those in the cytoplasmic leaflet. This idea is supported by the

average B-factors of the lipids, which tend to be lower for the

lipids in the extracellular leaflet, suggesting that their mobi-

lity is more restricted compared with those in the cytoplasmic

leaflet (Figure 3D and Supplementary Figure S3).

Lipid PE7 in AQP0EPL illustrates how the protein surface

defines the positions of the acyl chains of annular lipids

(Figure 4C). One acyl chain of PE7 lies in a groove on the

surface of AQP0 that guides it straight towards the centre of

the bilayer. The other acyl chain runs in between the bulky

side chains of Trp 10 and Phe 18 and then bends sharply to

evade the side chain of Leu 95, thus filling in a cleft formed by

Trp 10 on one side and Val 91 and Leu 95 on the other. The

two acyl chains of PE6 extend over the surface of this cleft,

completely covering it. Phosphatidylcholine 7, the lipid in

AQP0DMPC that corresponds to PE7 in AQP0EPL, adapts

differently to the protein surface (Figure 4D). Compared

with PE7, the glycerol backbone of PC7 is shifted by about

° away from the centre of the bilayer, which is probably

caused by the bulky side chain of Phe 14 adopting a different

conformation in AQP0DMPC. One of the acyl chains of PC7

follows the same groove in the AQP0 surface as the corre-

sponding acyl chain of PE7, extending straight to the centre of

the bilayer. The second acyl chain, however, follows a gap in

between the side chains of Trp 10 and Phe 14 in its different

position. It then immediately fills the cleft formed by residues

Trp 10, Phe 14, Val 91, and Leu 95. Thus, both PE7 and

PC7 adopt conformations that allow the acyl chains to fill in

the large hydrophobic pocket in the AQP0 surface, but in

different ways. To even out the protein surface is a crucial

function of annular lipids, because it ensures that the bilayer

forms a tight seal around the membrane protein and

preserves the separation of the cellular interior from the

external environment.

Discussion

Comparison of our new AQP0EPL structure with the pre-

viously determined AQP0DMPC structure shows that the an-

nular 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 and AQP0DMPC

structures, in particular the ones lining the water channel,

have nearly identical conformations. The virtually identical

channel structure in AQP0EPL and AQP0DMPC 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

(Figure 2A). 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 representa-

tion 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 hydro-

phobic mismatch, it has been proposed that either the lipids

Figure 4 EPL headgroups and acyl chains. (A) Lipid PE3 interacts

with AQP0 through a putative lipid-binding motif formed by Arg196

and Tyr105. (B) In AQP0DMPC the same residues do not interact with

the corresponding lipid, PC3. (C) Lipid PE7 in AQP0EPL bends to

follow the surface features of AQP0 and fills in a pocket formed by

residues Trp10, Val91, and Leu95. (D) The corresponding lipid in

AQP0DMPC, PC7, follows a different path but fills in the same pocket

in the AQP0 surface.

Interaction of AQPO with E. coli lipids

RK Hite et al

The EMBO Journal

VOL 29 | NO 10 | 2010

2010 European Molecular Biology Organization

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