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EMBO

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Principles of membrane protein interactions with

annular lipids deduced from aquaporin-0 2D

crystals

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits

distribution, and reproduction in any medium, provided the original author and source are credited. This license does not

permit commercial exploitation without specific permission.

Richard K Hite

1, Zongli Li1,2

and Thomas Walz

1,2,*

1Department of Cell Biology, Harvard Medical School, Boston, MA, USA

and

2Howard Hughes Medical Institute, Harvard Medical School,

Boston, MA, USA

have

previously

described

the

interactions

aquaporin-0 (AQP0) with dimyristoyl phosphatidylcholine

(DMPC) lipids. We have now determined the 2.5 A

° struc-

ture of AQP0 in two-dimensional (2D) crystals formed with

Escherichia coli polar lipids (EPLs), which differ from

DMPC both in headgroups and acyl chains. Comparison

of the two structures shows that AQP0 does not adapt to

the different length of the acyl chains in EPLs and that the

distance between the phosphodiester groups in the two

leaflets of the DMPC and EPL bilayers is almost identical.

The EPL headgroups interact differently with AQP0 than do

those of DMPC, but the acyl chains in the EPL and DMPC

bilayers occupy similar positions. The interactions of annu-

lar lipids with membrane proteins seem to be driven by the

propensity of the acyl chains to fill gaps in the protein

surface. Interactions of the lipid headgroups may be respon-

sible for the specific interactions found in tightly bound

lipids but seem to have a negligible effect on interactions

of generic annular lipids with membrane proteins.

The EMBO Journal (2010) 29, 16521658. doi:10.1038/

emboj.2010.68; Published online 13 April 2010

Subject Categories: membranes & transport; structural

biology

Keywords: electron crystallography; lens; lipidprotein inter-

actions; water channel

Introduction

How do membrane proteins interact with lipids? Spin-label-

ling and fluorescence-quenching studies have provided a

thermodynamic understanding of lipidprotein interactions,

but these methods do not allow a direct visualization of

individual interactions between a protein and a lipid. Most

of the available atomic resolution structural information on

lipidprotein interactions comes from lipids in crystal struc-

tures of membrane proteins in detergent micelles. A careful

analysis of all lipids bound to membrane proteins seen in

crystal structures deposited in the Protein Data Bank estab-

lished a lipid-binding motif. The motif consists of a positively

charged residue and a polar residue that specifically interact

with the negatively charged phosphodiester groups of the

lipids (Palsdottir and Hunte, 2004). Most of the co-crystal-

lized lipids originate from the donor membrane and must

have remained associated with the protein during solubiliza-

tion and purification to be incorporated in the three-dimen-

sional (3D) crystal. Therefore, lipids in crystal structures

must be strongly bound to the membrane proteins. These

lipids are a special case of `annular' lipids, the lipids in direct

contact with a membrane protein, because spin-labelling and

fluorescence-quenching studies demonstrated that most an-

nular lipids form only weak and non-specific interactions

with membrane proteins (Lee, 2003). Generic annular lipids

are thus typically lost during solubilization and/or purifica-

tion of membrane proteins and are usually not observed in

crystal structures.

The analysis of the lipids in 3D crystals also showed that

the lipid headgroups, and in particular the phosphodiester

groups, were tightly associated with the membrane proteins,

and thus were the best-ordered atoms of the lipids in these

structures (Palsdottir and Hunte, 2004). In contrast, density

for the lipid headgroups was poor in electron and X-ray

crystallographic density maps of bacteriorhodospsin (e.g.,

Grigorieff et al, 1996; Luecke et al, 1999), a light-driven

proton pump that forms crystalline arrays in the membrane

of Halobacterium salinarum, raising the question of what

general principles govern the interactions of membrane

proteins with their annular lipids.

Previously, the structure of the lens-specific water channel

aquaporin-0 (AQP0) was determined by electron crystallo-

graphy of double-layered, two-dimensional (2D) crystals,

revealing seven annular dimyristoyl phosphatidylcholine

(DMPC) molecules that surround each monomer and two

bulk lipids not in direct contact with AQP0 (Gonen et al,

2005). As with bacteriorhodopsin, examination of the elec-

tron crystallographic structure of AQP0 in the DMPC bilayer

revealed few favourable interactions of the lipid headgroups

with the protein surface (Hite et al, 2008). Although the

bacteriorhodopsin structures were obtained with the native

purple membrane lipids, AQP0 was completely delipidated

before reconstitution, and DMPC, a synthetic lipid, is not

found in biological membranes. Although the interactions

between bacteriorhodopsin and purple membrane lipids are

structurally and functionally best characterized, the lipid

protein interactions seen in the AQP0 structure may thus be

the most generic in nature. The situation of the lipids in AQP0

2D crystals is, however, special, because they are sandwiched

Received: 7 January 2010; accepted: 23 March 2010; published

online: 13 April 2010

*Corresponding author. Department of Cell Biology, Howard Hughes

Medical Institute, Harvard Medical School, 240 Longwood Avenue,

Boston, MA 02115, USA. Tel.: þ 1 617 432 4090;

Fax: þ 1 617 432 1144; E-mail: twalz@hms.harvard.edu

The EMBO Journal (2010) 29, 16521658

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