adjust their length to match the protein or that a-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
suggesting changes in protein structure. It is not clear, how-
ever, 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 (Figure 1D). 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
thickness of the bilayer formed by unsaturated lipids
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 sur-
rounded 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
(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 neigh-
bouring AQP0 tetramers in which no ordered lipid molecules
were found (asterisks in Figure 2A). 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 A
° , and DMPC,
33.6 A
between the C2 atoms of the glycerol of the lipids in the two
leaflets is larger for the shorter DMPC lipids, 31.2 A
° , than for
the longer EPLs, 27.0 A
the vertical position of the lipids is defined by the strong
charges of the phosphodiester groups that have to be posi-
tioned outside the hydrophobic belt of the membrane protein.
The glycerol backbone, however, which is largely hydropho-
bic 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 mem-
brane proteins may change their structure to adjust to
bilayers with a significantly different hydrophobic thickness,
it remains to be determined whether rigid membrane pro-
teins, 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 inter-
actions 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.
Materials and methods
Protein purification and crystallization
The core tissue of sheep lenses (Wolverine Packing Company,
Detroit, MI) was dissected away from the soft cortical tissue and
membranes were prepared as previously described (Gonen et al,
2004). Purified membranes were solubilized in 4% (w/v) octyl
glucoside in 10 mM Tris (pH 8.0) for 30 min at 221C. Insoluble
material was separated by centrifugation at 300 000 Â g. Solubilized
proteins were bound to a MonoQ column (GE Healthcare) and
eluted with 150 mM NaCl. Pooled fractions were run over a
Superose 12 (GE Healthcare) and eluted with 1.2% octyl glucoside
in 10 mM Tris (pH 8.0) and 150 mM NaCl. Purified AQP0 was
reconstituted into 2D crystals using E. coli polar lipids (Avanti Polar
Lipids) at a lipid-to-protein ratio of 0.25 (mg/mg) by dialysis against
10 mM MES (pH 6), 100 mM NaCl, and 50 mM MgCl2 at 371C for
1 week with daily buffer exchanges.
Data collection, data processing, model building,
and refinement
Specimens for cryo-electron microscopy were prepared using the
transferred into an FEI Tecnai Polara electron microscope operated
at an acceleration voltage of 300 kV. Low-dose electron diffraction
patterns were recorded at liquid helium temperature (
B6 K) with a
4 Â 4 K CCD camera (Gatan) and a camera length of 1.9 m. Electron
diffraction patterns were analysed and merged as described
2007) using as search model the 1.9-A
° electron crystallographic
PDB ID: 2B6O).
using 300 kV electron scattering factors. Protein residues 7226
were visible and were modelled and refined.
Building the lipids into the density map was complicated by the
structural heterogeneity of EPLs. All lipids were initially modelled
into the FoFc difference map with PE headgroups and short (
B810
carbon) acyl chains. Short acyl chains were used because some of
the chains were poorly defined in the initial difference map and
because several of the densities representing acyl chains were
branched. After a round of simulated annealing refinement, further
carbons were added to the acyl chains if additional density was
clearly visible in the composite omit map. For branched densities,
the strongest arm was chosen for model building. This process was
repeated iteratively until up to a maximum of 18 carbon atoms were
Interaction of AQPO with E. coli lipids
RK Hite et al
&
2010 European Molecular Biology Organization
The EMBO Journal
VOL 29 | NO 10 | 2010 1657