0	EMBOopen, otherFeat=[]-->, belongsTo=title
1	Principles of membrane protein interactions with, otherFeat=[u'b']-->, belongsTo=title
2	annular lipids deduced from aquaporin-0 2D, otherFeat=[u'b']-->, belongsTo=title
3	crystals, otherFeat=[u'b']-->, belongsTo=title
4	This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits, otherFeat=[]-->, belongsTo=parr
5	distribution, and reproduction in any medium, provided the original author and source are credited. This license does not, otherFeat=[]-->, belongsTo=parr
6	permit commercial exploitation without speci?c permission., otherFeat=[]-->, belongsTo=parr
7	Richard K Hite1, Zongli Li1,2, otherFeat=[u'b']-->, belongsTo=title
8	and Thomas Walz1,2,*, otherFeat=[u'b']-->, belongsTo=title
9	1Department of Cell Biology, Harvard Medical School, Boston, MA, USA, otherFeat=[]-->, belongsTo=parrnote
10	and 2Howard Hughes Medical Institute, Harvard Medical School,, otherFeat=[]-->, belongsTo=parrnote
11	Boston, MA, USA, otherFeat=[]-->, belongsTo=parrnote
12	We have previously described the interactions of, otherFeat=[]-->, belongsTo=parr
13	aquaporin-0 (AQP0) with dimyristoyl phosphatidylcholine, otherFeat=[]-->, belongsTo=parr
14	(DMPC) lipids. We have now determined the 2.5 A? struc-, otherFeat=[]-->, belongsTo=parr
15	ture of AQP0 in two-dimensional (2D) crystals formed with, otherFeat=[]-->, belongsTo=parr
16	Escherichia coli polar lipids (EPLs), which differ from, otherFeat=[]-->, belongsTo=parr
17	DMPC both in headgroups and acyl chains. Comparison, otherFeat=[]-->, belongsTo=parr
18	of the two structures shows that AQP0 does not adapt to, otherFeat=[]-->, belongsTo=parr
19	the different length of the acyl chains in EPLs and that the, otherFeat=[]-->, belongsTo=parr
20	distance between the phosphodiester groups in the two, otherFeat=[]-->, belongsTo=parr
21	lea?ets of the DMPC and EPL bilayers is almost identical., otherFeat=[]-->, belongsTo=parr
22	The EPL headgroups interact differently with AQP0 than do, otherFeat=[]-->, belongsTo=parr
23	those of DMPC, but the acyl chains in the EPL and DMPC, otherFeat=[]-->, belongsTo=parr
24	bilayers occupy similar positions. The interactions of annu-, otherFeat=[]-->, belongsTo=parr
25	lar lipids with membrane proteins seem to be driven by the, otherFeat=[]-->, belongsTo=parr
26	propensity of the acyl chains to ?ll gaps in the protein, otherFeat=[]-->, belongsTo=parr
27	surface. Interactions of the lipid headgroups may be respon-, otherFeat=[]-->, belongsTo=parr
28	sible for the speci?c interactions found in tightly bound, otherFeat=[]-->, belongsTo=parr
29	lipids but seem to have a negligible effect on interactions, otherFeat=[]-->, belongsTo=parr
30	of generic annular lipids with membrane proteins., otherFeat=[]-->, belongsTo=parr
31	The EMBO Journal (2010) 29, 1652?1658. doi:10.1038/, otherFeat=[]-->, belongsTo=parr
32	emboj.2010.68; Published online 13 April 2010, otherFeat=[]-->, belongsTo=parr
33	Subject Categories: membranes & transport; structural, otherFeat=[]-->, belongsTo=parr
34	biology, otherFeat=[]-->, belongsTo=parr
35	Keywords: electron crystallography; lens; lipid?protein inter-, otherFeat=[]-->, belongsTo=parr
36	actions; water channel, otherFeat=[]-->, belongsTo=parr
37	Introduction, otherFeat=[u'b']-->, belongsTo=title
38	How do membrane proteins interact with lipids? Spin-label-, otherFeat=[]-->, belongsTo=parr
39	ling and ?uorescence-quenching studies have provided a, otherFeat=[]-->, belongsTo=parr
40	thermodynamic understanding of lipid?protein interactions,, otherFeat=[]-->, belongsTo=parr
41	but these methods do not allow a direct visualization of, otherFeat=[]-->, belongsTo=parr
42	individual interactions between a protein and a lipid. Most, otherFeat=[]-->, belongsTo=parr
43	of the available atomic resolution structural information on, otherFeat=[]-->, belongsTo=parr
44	lipid?protein interactions comes from lipids in crystal struc-, otherFeat=[]-->, belongsTo=parr
45	tures of membrane proteins in detergent micelles. A careful, otherFeat=[]-->, belongsTo=parr
46	analysis of all lipids bound to membrane proteins seen in, otherFeat=[]-->, belongsTo=parr
47	crystal structures deposited in the Protein Data Bank estab-, otherFeat=[]-->, belongsTo=parr
48	lished a lipid-binding motif. The motif consists of a positively, otherFeat=[]-->, belongsTo=parr
49	charged residue and a polar residue that speci?cally interact, otherFeat=[]-->, belongsTo=parr
50	with the negatively charged phosphodiester groups of the, otherFeat=[]-->, belongsTo=parr
51	lipids (Palsdottir and Hunte, 2004). Most of the co-crystal-, otherFeat=[]-->, belongsTo=parr
52	lized lipids originate from the donor membrane and must, otherFeat=[]-->, belongsTo=parr
53	have remained associated with the protein during solubiliza-, otherFeat=[]-->, belongsTo=parr
54	tion and puri?cation to be incorporated in the three-dimen-, otherFeat=[]-->, belongsTo=parr
55	sional (3D) crystal. Therefore, lipids in crystal structures, otherFeat=[]-->, belongsTo=parr
56	must be strongly bound to the membrane proteins. These, otherFeat=[]-->, belongsTo=parr
57	lipids are a special case of ?annular? lipids, the lipids in direct, otherFeat=[]-->, belongsTo=parr
58	contact with a membrane protein, because spin-labelling and, otherFeat=[]-->, belongsTo=parr
59	?uorescence-quenching studies demonstrated that most an-, otherFeat=[]-->, belongsTo=parr
60	nular lipids form only weak and non-speci?c interactions, otherFeat=[]-->, belongsTo=parr
61	with membrane proteins (Lee, 2003). Generic annular lipids, otherFeat=[]-->, belongsTo=parr
62	are thus typically lost during solubilization and/or puri?ca-, otherFeat=[]-->, belongsTo=parr
63	tion of membrane proteins and are usually not observed in, otherFeat=[]-->, belongsTo=parr
64	crystal structures., otherFeat=[]-->, belongsTo=parr
65	The analysis of the lipids in 3D crystals also showed that, otherFeat=[]-->, belongsTo=parr
66	the lipid headgroups, and in particular the phosphodiester, otherFeat=[]-->, belongsTo=parr
67	groups, were tightly associated with the membrane proteins,, otherFeat=[]-->, belongsTo=parr
68	and thus were the best-ordered atoms of the lipids in these, otherFeat=[]-->, belongsTo=parr
69	structures (Palsdottir and Hunte, 2004). In contrast, density, otherFeat=[]-->, belongsTo=parr
70	for the lipid headgroups was poor in electron and X-ray, otherFeat=[]-->, belongsTo=parr
71	crystallographic density maps of bacteriorhodospsin (e.g.,, otherFeat=[]-->, belongsTo=parr
72	Grigorieff et al, 1996; Luecke et al, 1999), a light-driven, otherFeat=[]-->, belongsTo=parr
73	proton pump that forms crystalline arrays in the membrane, otherFeat=[]-->, belongsTo=parr
74	of Halobacterium salinarum, raising the question of what, otherFeat=[]-->, belongsTo=parr
75	general principles govern the interactions of membrane, otherFeat=[]-->, belongsTo=parr
76	proteins with their annular lipids., otherFeat=[]-->, belongsTo=parr
77	Previously, the structure of the lens-speci?c water channel, otherFeat=[]-->, belongsTo=parr
78	aquaporin-0 (AQP0) was determined by electron crystallo-, otherFeat=[]-->, belongsTo=parr
79	graphy of double-layered, two-dimensional (2D) crystals,, otherFeat=[]-->, belongsTo=parr
80	revealing seven annular dimyristoyl phosphatidylcholine, otherFeat=[]-->, belongsTo=parr
81	(DMPC) molecules that surround each monomer and two, otherFeat=[]-->, belongsTo=parr
82	bulk lipids not in direct contact with AQP0 (Gonen et al,, otherFeat=[]-->, belongsTo=parr
83	2005). As with bacteriorhodopsin, examination of the elec-, otherFeat=[]-->, belongsTo=parr
84	tron crystallographic structure of AQP0 in the DMPC bilayer, otherFeat=[]-->, belongsTo=parr
85	revealed few favourable interactions of the lipid headgroups, otherFeat=[]-->, belongsTo=parr
86	with the protein surface (Hite et al, 2008). Although the, otherFeat=[]-->, belongsTo=parr
87	bacteriorhodopsin structures were obtained with the native, otherFeat=[]-->, belongsTo=parr
88	purple membrane lipids, AQP0 was completely delipidated, otherFeat=[]-->, belongsTo=parr
89	before reconstitution, and DMPC, a synthetic lipid, is not, otherFeat=[]-->, belongsTo=parr
90	found in biological membranes. Although the interactions, otherFeat=[]-->, belongsTo=parr
91	between bacteriorhodopsin and purple membrane lipids are, otherFeat=[]-->, belongsTo=parr
92	structurally and functionally best characterized, the lipid?, otherFeat=[]-->, belongsTo=parr
93	protein interactions seen in the AQP0 structure may thus be, otherFeat=[]-->, belongsTo=parr
94	the most generic in nature. The situation of the lipids in AQP0, otherFeat=[]-->, belongsTo=parr
95	2D crystals is, however, special, because they are sandwiched, otherFeat=[]-->, belongsTo=parr
96	Received: 7 January 2010; accepted: 23 March 2010; published, otherFeat=[]-->, belongsTo=parrnote
97	online: 13 April 2010, otherFeat=[]-->, belongsTo=parrnote
98	*Corresponding author. Department of Cell Biology, Howard Hughes, otherFeat=[]-->, belongsTo=parrnote
99	Medical Institute, Harvard Medical School, 240 Longwood Avenue,, otherFeat=[]-->, belongsTo=parrnote
100	Boston, MA 02115, USA. Tel.: ? 1 617 432 4090;, otherFeat=[]-->, belongsTo=parrnote
101	Fax: ? 1 617 432 1144; E-mail: twalz@hms.harvard.edu, otherFeat=[]-->, belongsTo=parrnote
102	The EMBO Journal (2010) 29, 1652?1658 | & 2010 European Molecular Biology Organization | Some Rights Reserved 0261-4189/10, otherFeat=[]-->, belongsTo=parrnote
103	www.embojournal.org, otherFeat=[u'a']-->, belongsTo=parrnote
104	The EMBO Journal VOL 29 | NO 10 | 2010 & 2010 European Molecular Biology Organization, otherFeat=[]-->, belongsTo=parrnote
105	, otherFeat=[u'a']-->, belongsTo=title
106	EMBO, otherFeat=['U', u'a']-->, belongsTo=title
107	, otherFeat=[u'a']-->, belongsTo=title
108	THE, otherFeat=[u'a']-->, belongsTo=title
109	EMBO, otherFeat=['U', u'a']-->, belongsTo=title
110	JOURNAL, otherFeat=[u'a']-->, belongsTo=title
111	THE, otherFeat=[u'a']-->, belongsTo=title
112	EMBO, otherFeat=['U', u'a']-->, belongsTo=title
113	JOURNAL, otherFeat=[u'a']-->, belongsTo=title
114	1652, otherFeat=[u'b']-->, belongsTo=nota_cab_pie
115	in between two AQP0 tetramers, which is not typically the, otherFeat=[]-->, belongsTo=parr
116	case for lipids surrounding membrane proteins in biological, otherFeat=[]-->, belongsTo=parr
117	membranes., otherFeat=[]-->, belongsTo=parr
118	The tight positioning of the lipids in AQP0 crystals reduced, otherFeat=[]-->, belongsTo=parr
119	their mobility and rendered them visible in the density maps,, otherFeat=[]-->, belongsTo=parr
120	making it possible to describe the lipid?protein interactions., otherFeat=[]-->, belongsTo=parr
121	If AQP0 2D crystals could be grown with other lipids, such, otherFeat=[]-->, belongsTo=parr
122	crystals would provide an opportunity to study how a mem-, otherFeat=[]-->, belongsTo=parr
123	brane protein interacts with different lipids in a near-native, otherFeat=[]-->, belongsTo=parr
124	environment. While formation of high-quality 2D crystals, otherFeat=[]-->, belongsTo=parr
125	usually requires the use of a speci?c lipid, aquaporins can, otherFeat=[]-->, belongsTo=parr
126	often form 2D crystals with a variety of lipids. AQP1, for, otherFeat=[]-->, belongsTo=parr
127	example, formed large, well-ordered 2D crystals with three, otherFeat=[]-->, belongsTo=parr
128	different lipids (Jap and Li, 1995; Murata et al, 2000; Ren, otherFeat=[]-->, belongsTo=parr
129	et al, 2000). In the case of AQP0, 2D crystallization screens, otherFeat=[]-->, belongsTo=parr
130	showed that, in addition to DMPC, AQP0 also forms 2D, otherFeat=[]-->, belongsTo=parr
131	crystals with E. coli polar lipids (EPLs). E. coli polar lipids, otherFeat=[]-->, belongsTo=parr
132	differ from DMPC in headgroup chemistry, as well as acyl, otherFeat=[]-->, belongsTo=parr
133	chain length and saturation. The headgroups of EPLs are a, otherFeat=[]-->, belongsTo=parr
134	mixture of phosphatidylethanolamine (PE), phosphatidylgly-, otherFeat=[]-->, belongsTo=parr
135	cerol (PG) and cardiolipin (CL) rather than the pure phos-, otherFeat=[]-->, belongsTo=parr
136	phatidylcholine (PC) headgroup of DMPC. The acyl chains of, otherFeat=[]-->, belongsTo=parr
137	DMPC are two saturated 14-carbon acyl chains, whereas the, otherFeat=[]-->, belongsTo=parr
138	average length of the acyl chains of EPLs is 16 carbon atoms,, otherFeat=[]-->, belongsTo=parr
139	and approximately 55% of the acyl chains are unsaturated, otherFeat=[]-->, belongsTo=parr
140	(Lugtenberg and Peters, 1976). We have now optimized the, otherFeat=[]-->, belongsTo=parr
141	quality of the AQP0 2D crystals formed with EPLs and, otherFeat=[]-->, belongsTo=parr
142	produced a density map at 2.5 A? resolution. As with the, otherFeat=[]-->, belongsTo=parr
143	previous density map produced with 2D crystals formed, otherFeat=[]-->, belongsTo=parr
144	with DMPC, the current density map revealed the seven, otherFeat=[]-->, belongsTo=parr
145	annular lipids, allowing us to compare the interactions of, otherFeat=[]-->, belongsTo=parr
146	AQP0 with two very different lipids, DMPC and EPLs., otherFeat=[]-->, belongsTo=parr
147	Results, otherFeat=[u'b']-->, belongsTo=title
148	Structure of AQP0 in DMPC and EPL bilayers, otherFeat=[u'b']-->, belongsTo=parr
149	Well-ordered, double-layered 2D crystals of AQP0 formed, otherFeat=[]-->, belongsTo=parr
150	with EPLs at a lipid-to-protein ratio of 0.25 (mg/mg;, otherFeat=[]-->, belongsTo=parr
151	Figure 1A). These crystals were used to record electron, otherFeat=[]-->, belongsTo=parr
152	diffraction patterns at liquid helium temperature (B6 K)., otherFeat=[]-->, belongsTo=parr
153	Diffraction patterns from untilted crystals showed re?ections, otherFeat=[]-->, belongsTo=parr
154	beyond 1.9 A? (Supplementary Figure S1), but the resolution, otherFeat=[]-->, belongsTo=parr
155	was more limited in diffraction patterns of highly tilted, otherFeat=[]-->, belongsTo=parr
156	crystals (Supplementary Figure S2). After merging 281 dif-, otherFeat=[]-->, belongsTo=parr
157	fraction patterns and phasing by molecular replacement, we, otherFeat=[]-->, belongsTo=parr
158	produced a density map at 2.5 A? resolution (Figure 1B) and, otherFeat=[]-->, belongsTo=parr
159	modelled and re?ned the structure of AQP0 (residues 7?226), otherFeat=[]-->, belongsTo=parr
160	in the membrane junction (Figure 1C; Table I). Residues 1?6, otherFeat=[]-->, belongsTo=parr
161	and 227?263, which include the C-terminal helix modelled in, otherFeat=[]-->, belongsTo=parr
162	the previous structure of AQP0 in a DMPC bilayer (Gonen, otherFeat=[]-->, belongsTo=parr
163	et al, 2005), did not show clear density in the map and were, otherFeat=[]-->, belongsTo=parr
164	therefore excluded from the model of AQP0 in the EPL, otherFeat=[]-->, belongsTo=parr
165	bilayer., otherFeat=[]-->, belongsTo=parr
166	Other than the C-terminal helix, which was disordered and, otherFeat=[]-->, belongsTo=parr
167	therefore could not be modelled, the structure of AQP0 in the, otherFeat=[]-->, belongsTo=parr
168	EPL bilayer (AQP0EPL) is virtually identical with its structure, otherFeat=[]-->, belongsTo=parr
169	in the DMPC bilayer (AQP0DMPC ; Figure 1D). The r.m.s.d., otherFeat=[]-->, belongsTo=parr
170	values between all modelled backbone atoms is 0.48 A? ,, otherFeat=[]-->, belongsTo=parr
171	and 0.4 A? if only the transmembrane domains are compared., otherFeat=[]-->, belongsTo=parr
172	The pore-lining residues in AQP0EPL and AQP0DMPC are also, otherFeat=[]-->, belongsTo=parr
173	essentially the same, and both structures show three water, otherFeat=[]-->, belongsTo=parr
174	molecules in the centre of the channel at the same positions, otherFeat=[]-->, belongsTo=parr
175	(Figure 1E)., otherFeat=[u'a']-->, belongsTo=parr
176	Modelling the EPL bilayer, otherFeat=[u'b']-->, belongsTo=parr
177	In addition to the protein, the density map also allowed us to, otherFeat=[]-->, belongsTo=parr
178	build all seven annular lipids surrounding each AQP0 mono-, otherFeat=[]-->, belongsTo=parr
179	mer, but not the lipids in the central area between four, otherFeat=[]-->, belongsTo=parr
180	adjacent tetramers (asterisks in Figure 2A). The acyl chains, otherFeat=[]-->, belongsTo=parr
181	of the annular lipids were initially modelled as 10 carbon, otherFeat=[]-->, belongsTo=parr
182	chains and then extended to occupy the maximal length that, otherFeat=[]-->, belongsTo=parr
183	was clearly resolved in the density map. In the initial density, otherFeat=[]-->, belongsTo=parr
184	map, densities representing the acyl chains were often, otherFeat=[]-->, belongsTo=parr
185	branched, indicating that they can adopt multiple conforma-, otherFeat=[]-->, belongsTo=parr
186	tions. As the data were insuf?cient for the re?nement of, otherFeat=[]-->, belongsTo=parr
187	alternative conformations, we chose to build each acyl chain, otherFeat=[]-->, belongsTo=parr
188	into the strongest density, representing the predominant, otherFeat=[]-->, belongsTo=parr
189	lipid conformation. After several rounds of re?nement, the, otherFeat=[]-->, belongsTo=parr
190	Figure 1 Double-layered 2D crystals of AQP0 in Escherichia coli polar lipids (EPLs). (A) Representative AQP0 2D crystal formed with EPLs in, otherFeat=[]-->, belongsTo=fig_caption
191	negative stain. Scale bar: 1 mm. (B) Region of the ?nal 2Fo ?Fc map re?ned to 2.5 A? resolution showing pore-lining residues and a water, otherFeat=[]-->, belongsTo=fig_caption
192	molecule. (C) Atomic model of the AQP0 membrane junction. (D) Overlay of the AQP0EPL (gold) and AQP0DMPC (light blue) structures. (E) The, otherFeat=[]-->, belongsTo=fig_caption
193	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, otherFeat=[]-->, belongsTo=fig_caption
194	previously seen in AQP0DMPC (blue). The 2Fo?Fc density map for the water molecules is shown at a contouring level of 1s (blue wire mesh)., otherFeat=[]-->, belongsTo=fig_caption
195	Interaction of AQPO with E. coli lipids, otherFeat=[]-->, belongsTo=nota_cab_pie
196	RK Hite et al, otherFeat=[]-->, belongsTo=nota_cab_pie
197	& 2010 European Molecular Biology Organization, otherFeat=[]-->, belongsTo=nota_cab_pie
198	The EMBO Journal, otherFeat=[]-->, belongsTo=nota_cab_pie
199	VOL 29, otherFeat=['U']-->, belongsTo=nota_cab_pie
200	|, otherFeat=[]-->, belongsTo=nota_cab_pie
201	NO 10, otherFeat=['U']-->, belongsTo=nota_cab_pie
202	|, otherFeat=[]-->, belongsTo=nota_cab_pie
203	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
204	1653, otherFeat=[u'b']-->, belongsTo=nota_cab_pie
205	branches disappeared, and all acyl chains were represented, otherFeat=[]-->, belongsTo=parr
206	by a single, mostly continuous density., otherFeat=[]-->, belongsTo=parr
207	Approximately 55% of the acyl chains of EPLs contain an, otherFeat=[]-->, belongsTo=parr
208	unsaturated bond, with the two most abundant species being, otherFeat=[]-->, belongsTo=parr
209	16c1:9 and 18c1:11 (Lugtenberg and Peters, 1976). Although, otherFeat=[]-->, belongsTo=parr
210	some lipids showed kinks that could indicate the presence of, otherFeat=[]-->, belongsTo=parr
211	a double bond, due to the heterogeneity of the acyl chains in, otherFeat=[]-->, belongsTo=parr
212	EPLs, we modelled all acyl chains as being fully saturated., otherFeat=[]-->, belongsTo=parr
213	Escherichia coli polar lipids are a mixture of three different, otherFeat=[]-->, belongsTo=parr
214	headgroups (67% PE, 23% PG and 10% CL lipids; Oursel et al, otherFeat=[]-->, belongsTo=parr
215	(2007)) with PE being by far the most abundant headgroup., otherFeat=[]-->, belongsTo=parr
216	We therefore initially modelled all headgroups as PE. After, otherFeat=[]-->, belongsTo=parr
217	re?nement, we found no evidence in the density map, which, otherFeat=[]-->, belongsTo=parr
218	suggested that any of the lipid positions is preferentially, otherFeat=[]-->, belongsTo=parr
219	occupied by a lipid with a particular headgroup. Our ?nal, otherFeat=[]-->, belongsTo=parr
220	model of the EPL bilayer thus contains seven PE molecules, otherFeat=[]-->, belongsTo=parr
221	with saturated acyl chains ranging from 5 to 17 carbon atoms, otherFeat=[]-->, belongsTo=parr
222	(Figure 2B)., otherFeat=[u'a']-->, belongsTo=parr
223	Organization of the EPL and DMPC bilayers surrounding, otherFeat=[u'b']-->, belongsTo=parr
224	AQP0, otherFeat=[u'b']-->, belongsTo=parr
225	The EPL and DMPC bilayers surrounding AQP0 are remark-, otherFeat=[]-->, belongsTo=parr
226	ably similar (Figure 2B and C). The two bilayers contain the, otherFeat=[]-->, belongsTo=parr
227	same number of lipids at comparable positions (Figure 3C), otherFeat=[]-->, belongsTo=parr
228	and have almost the same thickness (the average distance, otherFeat=[]-->, belongsTo=parr
229	between the phosphodiester groups in the two lea?ets is, otherFeat=[]-->, belongsTo=parr
230	31.9 A? for the EPL bilayer and 33.6 A? for the DMPC bilayer;, otherFeat=[]-->, belongsTo=parr
231	Figure 3A). As acyl chains of EPLs are on average longer than, otherFeat=[]-->, belongsTo=parr
232	those of DMPC (16 versus 14 carbon atoms), this ?nding, otherFeat=[]-->, belongsTo=parr
233	raises the question how the longer EPL acyl chains are, otherFeat=[]-->, belongsTo=parr
234	accommodated. Unexpectedly, despite the longer acyl chains, otherFeat=[]-->, belongsTo=parr
235	of EPLs, the average distance between the C2 atoms of, otherFeat=[]-->, belongsTo=parr
236	glycerols in the two lea?ets of the EPL bilayer, 27.0 A? , is, otherFeat=[]-->, belongsTo=parr
237	smaller than the corresponding average distance in the, otherFeat=[]-->, belongsTo=parr
238	DMPC bilayer, 31.2 A? (Figure 3B). Further comparison of, otherFeat=[]-->, belongsTo=parr
239	the AQP0 EPL and AQP0DMPC structures reveals that the, otherFeat=[]-->, belongsTo=parr
240	DMPC molecules cover less surface area on AQP0 than EPLs, otherFeat=[]-->, belongsTo=parr
241	(Figures 2B and C). Indeed, the DMPC bilayer leaves areas of, otherFeat=[]-->, belongsTo=parr
242	the hydrophobic surface of AQP0 uncovered (Figure 2C, e.g., otherFeat=[]-->, belongsTo=parr
243	area in between PC3 and PC4 of the extracellular lea?et and, otherFeat=[]-->, belongsTo=parr
244	Figure 2 The EPL bilayer. (A) Top view of the AQP0 2D crystal, otherFeat=[]-->, belongsTo=fig_caption
245	showing AQP0 tetramers (gold) and the surrounding EPLs (red). (B,, otherFeat=[]-->, belongsTo=fig_caption
246	C) The seven (B) annular EPLs and (C) DMPC molecules surround-, otherFeat=[]-->, belongsTo=fig_caption
247	ing an AQP0 monomer. As lipids are sandwiched between two, otherFeat=[]-->, belongsTo=fig_caption
248	adjacent AQP0 subunits, their positions relative to both AQP0, otherFeat=[]-->, belongsTo=fig_caption
249	subunits are shown., otherFeat=[]-->, belongsTo=fig_caption
250	Table I Electron crystallographic data, otherFeat=[]-->, belongsTo=parrnote
251	Two-dimensional crystals, otherFeat=[]-->, belongsTo=parrnote
252	Layer group p422, otherFeat=[]-->, belongsTo=parrnote
253	Unit cell a ? b ? 65.5 A?, otherFeat=[]-->, belongsTo=parrnote
254	Thickness (assumed) 200 A?, otherFeat=[]-->, belongsTo=parrnote
255	Electron diffraction, otherFeat=[]-->, belongsTo=parrnote
256	Number of patterns merged 281 (01: 11; 201: 22;, otherFeat=[]-->, belongsTo=parrnote
257	451: 63; 601: 108; 701: 77), otherFeat=[]-->, belongsTo=parrnote
258	Resolution limit for merging 2.3 A?, otherFeat=[]-->, belongsTo=parrnote
259	RFriedel 18.9%, otherFeat=[]-->, belongsTo=parrnote
260	RMerge 22.6%, otherFeat=[]-->, belongsTo=parrnote
261	Observed amplitudes to 2.5 A? 129 893, otherFeat=[]-->, belongsTo=parrnote
262	Unique re?ections 14 417, otherFeat=[]-->, belongsTo=parrnote
263	Maximum tilt angle 74.21, otherFeat=[]-->, belongsTo=parrnote
264	Fourier space sampled 92.3% (83.5% at 2.6?2.5 A? ), otherFeat=[]-->, belongsTo=parrnote
265	Multiplicity 8.1 (4.0 at 2.6?2.5 A? ), otherFeat=[]-->, belongsTo=parrnote
266	Crystallographic re?nement (10.0?2.5 A?), otherFeat=[]-->, belongsTo=parrnote
267	Resolution limit for re?nement 2.5 A?, otherFeat=[]-->, belongsTo=parrnote
268	Crystallographic R factor 24.98%, otherFeat=[]-->, belongsTo=parrnote
269	Free R factor 28.43%, otherFeat=[]-->, belongsTo=parrnote
270	Re?ections in working/test set 12 801/1453, otherFeat=[]-->, belongsTo=parrnote
271	Non-hydrogen protein atoms 1668, otherFeat=[]-->, belongsTo=parrnote
272	Non-hydrogen lipid atoms 273, otherFeat=[]-->, belongsTo=parrnote
273	Solvent molecules 8, otherFeat=[]-->, belongsTo=parrnote
274	Average protein B factor (A? 2) 42.3, otherFeat=[]-->, belongsTo=parrnote
275	Average lipid B factor (A? 2) 88.0, otherFeat=[]-->, belongsTo=parrnote
276	Ramachandran plot (%) 98.4/1.6/0.0 (allowed;, otherFeat=[]-->, belongsTo=parrnote
277	generous; disallowed), otherFeat=[]-->, belongsTo=parrnote
278	Rfree is calculated from a randomly chosen 10% of re?ections, and, otherFeat=[]-->, belongsTo=parrnote
279	Rcryst is calculated over the remaining 90% of re?ections., otherFeat=[]-->, belongsTo=parrnote
280	Interaction of AQPO with E. coli lipids, otherFeat=[]-->, belongsTo=nota_cab_pie
281	RK Hite et al, otherFeat=[]-->, belongsTo=nota_cab_pie
282	The EMBO Journal, otherFeat=[]-->, belongsTo=nota_cab_pie
283	VOL 29, otherFeat=['U']-->, belongsTo=nota_cab_pie
284	|, otherFeat=[]-->, belongsTo=nota_cab_pie
285	NO 10, otherFeat=['U']-->, belongsTo=nota_cab_pie
286	|, otherFeat=[]-->, belongsTo=nota_cab_pie
287	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
288	& 2010 European Molecular Biology Organization, otherFeat=[]-->, belongsTo=nota_cab_pie
289	1654, otherFeat=[u'b']-->, belongsTo=nota_cab_pie
290	PC7 of the cytoplasmic lea?et), suggesting that 14-carbon acyl, otherFeat=[]-->, belongsTo=parr
291	chains are close to the minimum needed to saturate the, otherFeat=[]-->, belongsTo=parr
292	hydrophobic belt of AQP0. Furthermore, unlike the lipids in, otherFeat=[]-->, belongsTo=parr
293	the DMPC bilayer, several of the longer EPL acyl chains, otherFeat=[]-->, belongsTo=parr
294	interdigitate in the middle of the bilayer, ?lling gaps between, otherFeat=[]-->, belongsTo=parr
295	acyl chains in the opposite lea?et (Figure 2B; e.g. one acyl, otherFeat=[]-->, belongsTo=parr
296	chain of PE7 inserts between the two acyl chains of PE3 and, otherFeat=[]-->, belongsTo=parr
297	the other acyl chain of PE7 inserts between the two acyl, otherFeat=[]-->, belongsTo=parr
298	chains of PE4). Other acyl chains bend sharply as they, otherFeat=[]-->, belongsTo=parr
299	approach the midpoint of the bilayer and then extend, otherFeat=[]-->, belongsTo=parr
300	parallel to the membrane plane (Figure 2B; e.g. acyl chains, otherFeat=[]-->, belongsTo=parr
301	of PE4, PE6, and PE7). Such bending may be facilitated by, otherFeat=[]-->, belongsTo=parr
302	unsaturated bonds present in acyl chains of EPLs, which have, otherFeat=[]-->, belongsTo=parr
303	previously been proposed to explain the bent conformations, otherFeat=[]-->, belongsTo=parr
304	seen in lipids associated with cytochrome b?c1 complexes, otherFeat=[]-->, belongsTo=parr
305	(Palsdottir and Hunte, 2004). Indeed, the positions of the, otherFeat=[]-->, belongsTo=parr
306	kinks seen in the acyl chains of lipids PE3, PE4, and PE6, otherFeat=[]-->, belongsTo=parr
307	occur close to the positions of the double bonds in the most, otherFeat=[]-->, belongsTo=parr
308	abundant unsaturated acyl chains of EPLs, 16c1:9 and 18c1:11,, otherFeat=[]-->, belongsTo=parr
309	whereas the kink in the acyl chain of PE7 is located between, otherFeat=[]-->, belongsTo=parr
310	C6 and C7., otherFeat=[]-->, belongsTo=parr
311	Interaction of the lipid headgroups with AQP0, otherFeat=[u'b']-->, belongsTo=parr
312	The AQP0EPL and AQP0DMPC structures make it possible to, otherFeat=[]-->, belongsTo=parr
313	analyse the interactions of AQP0 with different lipids., otherFeat=[]-->, belongsTo=parr
314	Although the corresponding lipids in the two bilayers are, otherFeat=[]-->, belongsTo=parr
315	at very similar positions (Figure 3C), all the headgroups of, otherFeat=[]-->, belongsTo=parr
316	the corresponding lipids in the two bilayers adopt different, otherFeat=[]-->, belongsTo=parr
317	conformations. Furthermore, none of the DMPC lipids in the, otherFeat=[]-->, belongsTo=parr
318	AQP0DMPC structure interacted with AQP0 through a lipid-, otherFeat=[]-->, belongsTo=parr
319	binding motif (Gonen et al, 2005), and only one lipid, PE3, in, otherFeat=[]-->, belongsTo=parr
320	the AQP0EPL structure is in an environment consistent with a, otherFeat=[]-->, belongsTo=parr
321	lipid-binding motif: the phosphodiester group of PE3 interacts, otherFeat=[]-->, belongsTo=parr
322	with a positively charged residue, Arg 196, and a polar residue,, otherFeat=[]-->, belongsTo=parr
323	Tyr 105 (Figure 4A). In PC3, the corresponding lipid in the, otherFeat=[]-->, belongsTo=parr
324	AQP0DMPC structure (Figure 4B), the glycerol backbone is, otherFeat=[]-->, belongsTo=parr
325	shifted by B3.4 A? closer to the centre of the lipid bilayer., otherFeat=[]-->, belongsTo=parr
326	When Hunte and coworkers identi?ed the lipid-binding motif,, otherFeat=[]-->, belongsTo=parr
327	they noted that it did not seem to apply to PC lipids and, otherFeat=[]-->, belongsTo=parr
328	suggested that this may be due to the large choline headgroup, otherFeat=[]-->, belongsTo=parr
329	that could cause steric clashes (Palsdottir and Hunte, 2004). We, otherFeat=[]-->, belongsTo=parr
330	were therefore unsure whether the changes in the conforma-, otherFeat=[]-->, belongsTo=parr
331	tion of PE3 with respect to PC3 were the result of the presence, otherFeat=[]-->, belongsTo=parr
332	of a lipid-binding motif. When we attempted to reposition PC3, otherFeat=[]-->, belongsTo=parr
333	to a location more similar to PE3, we found that there was, otherFeat=[]-->, belongsTo=parr
334	suf?cient space for a choline headgroup to occupy the same, otherFeat=[]-->, belongsTo=parr
335	space as the ethanolamine headgroup of PE3. As the choline, otherFeat=[]-->, belongsTo=parr
336	headgroup of PC3 does not occupy this position, even though it, otherFeat=[]-->, belongsTo=parr
337	would be sterically possible, and therefore does not interact, otherFeat=[]-->, belongsTo=parr
338	with AQP0 through Arg 196 and Tyr 105, we conclude that, otherFeat=[]-->, belongsTo=parr
339	these two residues do not constitute a true lipid-binding motif., otherFeat=[]-->, belongsTo=parr
340	Together, these observations suggest that the exact chemical, otherFeat=[]-->, belongsTo=parr
341	identities of the phospholipid headgroups have a negligible role, otherFeat=[]-->, belongsTo=parr
342	in the interaction of annular lipids with membrane proteins,, otherFeat=[]-->, belongsTo=parr
343	corroborating previous results obtained by spin-labelling, otherFeat=[]-->, belongsTo=parr
344	studies (Lee, 2003)., otherFeat=[]-->, belongsTo=parr
345	Figure 3 Comparisons between the EPL and DMPC bilayers. (A) The average distance between the phosphodiester groups in the two lea?ets of, otherFeat=[]-->, belongsTo=fig_caption
346	the bilayer formed by EPLs (31.9 A? ) is very similar to that between the phosphodiester groups in the DMPC bilayers (33.6 A?). Phosphorous, otherFeat=[]-->, belongsTo=fig_caption
347	atoms are shown in orange. Red shading represents the region between the most distantly located phosphorous atoms in each lea?et., otherFeat=[]-->, belongsTo=fig_caption
348	(B) Despite the longer acyl chains of EPLs, the average distance between the C2 atoms of the lipids? glycerol backbones in the two lea?ets of the, otherFeat=[]-->, belongsTo=fig_caption
349	bilayer formed by EPLs (27.0 A? ) is shorter than that between the C2 atoms in the DMPC bilayers (31.2 A?). The glycerol groups are shown in, otherFeat=[]-->, belongsTo=fig_caption
350	green. The green shading represents the region between the two most distantly located C2 atoms in each lea?et. (C) Overlay of the lipids seen, otherFeat=[]-->, belongsTo=fig_caption
351	in the AQP0EPL (red) and AQP0DMPC (blue) structures. (D) Same as in (C) with the lipids coloured according to their B-factors. Colour coding:, otherFeat=[]-->, belongsTo=fig_caption
352	intense blue, B-factors below 75 A? 2; pale blue, B-factors between 75 and 94 A? 2 ; pale red, B-factors between 95 and 114 A? 2; intense red, B-factors, otherFeat=[]-->, belongsTo=fig_caption
353	above 115 A? 2., otherFeat=[]-->, belongsTo=fig_caption
354	Interaction of AQPO with E. coli lipids, otherFeat=[]-->, belongsTo=nota_cab_pie
355	RK Hite et al, otherFeat=[]-->, belongsTo=nota_cab_pie
356	& 2010 European Molecular Biology Organization, otherFeat=[]-->, belongsTo=nota_cab_pie
357	The EMBO Journal, otherFeat=[]-->, belongsTo=nota_cab_pie
358	VOL 29, otherFeat=['U']-->, belongsTo=nota_cab_pie
359	|, otherFeat=[]-->, belongsTo=nota_cab_pie
360	NO 10, otherFeat=['U']-->, belongsTo=nota_cab_pie
361	|, otherFeat=[]-->, belongsTo=nota_cab_pie
362	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
363	1655, otherFeat=[u'b']-->, belongsTo=nota_cab_pie
364	Interaction of the lipid acyl chains with AQP0, otherFeat=[u'b']-->, belongsTo=parr
365	In contrast to the headgroups, acyl chains seem to be the, otherFeat=[]-->, belongsTo=parr
366	crucial element guiding the interaction of annular lipids with, otherFeat=[]-->, belongsTo=parr
367	the protein. Comparison of the lipids in AQP0EPL and, otherFeat=[]-->, belongsTo=parr
368	AQP0DMPC reveals that the acyl chains in the extracellular, otherFeat=[]-->, belongsTo=parr
369	lea?et occupy quite similar positions. The acyl chains of PE3, otherFeat=[]-->, belongsTo=parr
370	and PE4 extend along the same paths as those of the, otherFeat=[]-->, belongsTo=parr
371	corresponding lipids, PC3, and PC4. The two acyl chains of, otherFeat=[]-->, belongsTo=parr
372	PE2 are in similar positions as those occupied by one of the, otherFeat=[]-->, belongsTo=parr
373	acyl chains of PC1 and one of the acyl chains of PC2, otherFeat=[]-->, belongsTo=parr
374	(Figure 3C and Supplementary Video S1)., otherFeat=[]-->, belongsTo=parr
375	The positions of the acyl chains in the cytoplasmic lea?et, otherFeat=[]-->, belongsTo=parr
376	vary to a greater degree between AQP0 EPL and AQP0DMPC, otherFeat=[]-->, belongsTo=parr
377	than those of the acyl chains in the extracellular lea?et, otherFeat=[]-->, belongsTo=parr
378	(Figure 3C). The gap between two adjacent AQP0 tetramers, otherFeat=[]-->, belongsTo=parr
379	is narrower on the extracellular side of the bilayer, which may, otherFeat=[]-->, belongsTo=parr
380	force the lipids in this lea?et into more de?ned positions than, otherFeat=[]-->, belongsTo=parr
381	those in the cytoplasmic lea?et. This idea is supported by the, otherFeat=[]-->, belongsTo=parr
382	average B-factors of the lipids, which tend to be lower for the, otherFeat=[]-->, belongsTo=parr
383	lipids in the extracellular lea?et, suggesting that their mobi-, otherFeat=[]-->, belongsTo=parr
384	lity is more restricted compared with those in the cytoplasmic, otherFeat=[]-->, belongsTo=parr
385	lea?et (Figure 3D and Supplementary Figure S3)., otherFeat=[]-->, belongsTo=parr
386	Lipid PE7 in AQP0EPL illustrates how the protein surface, otherFeat=[]-->, belongsTo=parr
387	de?nes the positions of the acyl chains of annular lipids, otherFeat=[]-->, belongsTo=parr
388	(Figure 4C). One acyl chain of PE7 lies in a groove on the, otherFeat=[]-->, belongsTo=parr
389	surface of AQP0 that guides it straight towards the centre of, otherFeat=[]-->, belongsTo=parr
390	the bilayer. The other acyl chain runs in between the bulky, otherFeat=[]-->, belongsTo=parr
391	side chains of Trp 10 and Phe 18 and then bends sharply to, otherFeat=[]-->, belongsTo=parr
392	evade the side chain of Leu 95, thus ?lling in a cleft formed by, otherFeat=[]-->, belongsTo=parr
393	Trp 10 on one side and Val 91 and Leu 95 on the other. The, otherFeat=[]-->, belongsTo=parr
394	two acyl chains of PE6 extend over the surface of this cleft,, otherFeat=[]-->, belongsTo=parr
395	completely covering it. Phosphatidylcholine 7, the lipid in, otherFeat=[]-->, belongsTo=parr
396	AQP0DMPC that corresponds to PE7 in AQP0EPL , adapts, otherFeat=[]-->, belongsTo=parr
397	differently to the protein surface (Figure 4D). Compared, otherFeat=[]-->, belongsTo=parr
398	with PE7, the glycerol backbone of PC7 is shifted by about, otherFeat=[]-->, belongsTo=parr
399	3 A? away from the centre of the bilayer, which is probably, otherFeat=[]-->, belongsTo=parr
400	caused by the bulky side chain of Phe 14 adopting a different, otherFeat=[]-->, belongsTo=parr
401	conformation in AQP0DMPC. One of the acyl chains of PC7, otherFeat=[]-->, belongsTo=parr
402	follows the same groove in the AQP0 surface as the corre-, otherFeat=[]-->, belongsTo=parr
403	sponding acyl chain of PE7, extending straight to the centre of, otherFeat=[]-->, belongsTo=parr
404	the bilayer. The second acyl chain, however, follows a gap in, otherFeat=[]-->, belongsTo=parr
405	between the side chains of Trp 10 and Phe 14 in its different, otherFeat=[]-->, belongsTo=parr
406	position. It then immediately ?lls the cleft formed by residues, otherFeat=[]-->, belongsTo=parr
407	Trp 10, Phe 14, Val 91, and Leu 95. Thus, both PE7 and, otherFeat=[]-->, belongsTo=parr
408	PC7 adopt conformations that allow the acyl chains to ?ll in, otherFeat=[]-->, belongsTo=parr
409	the large hydrophobic pocket in the AQP0 surface, but in, otherFeat=[]-->, belongsTo=parr
410	different ways. To even out the protein surface is a crucial, otherFeat=[]-->, belongsTo=parr
411	function of annular lipids, because it ensures that the bilayer, otherFeat=[]-->, belongsTo=parr
412	forms a tight seal around the membrane protein and, otherFeat=[]-->, belongsTo=parr
413	preserves the separation of the cellular interior from the, otherFeat=[]-->, belongsTo=parr
414	external environment., otherFeat=[]-->, belongsTo=parr
415	Discussion, otherFeat=[u'b']-->, belongsTo=title
416	Comparison of our new AQP0EPL structure with the pre-, otherFeat=[]-->, belongsTo=parr
417	viously determined AQP0DMPC structure shows that the an-, otherFeat=[]-->, belongsTo=parr
418	nular lipids studied here have very little in?uence on the, otherFeat=[]-->, belongsTo=parr
419	structure of AQP0. With only a few exceptions, such as Phe, otherFeat=[]-->, belongsTo=parr
420	14, the amino-acid residues in the AQP0EPL and AQP0DMPC, otherFeat=[]-->, belongsTo=parr
421	structures, in particular the ones lining the water channel,, otherFeat=[]-->, belongsTo=parr
422	have nearly identical conformations. The virtually identical, otherFeat=[]-->, belongsTo=parr
423	channel structure in AQP0EPL and AQP0DMPC is consistent, otherFeat=[]-->, belongsTo=parr
424	with a previous study that showed that the lipid environment, otherFeat=[]-->, belongsTo=parr
425	did not affect water conduction by AQP1 (Zeidel et al, 1994),, otherFeat=[]-->, belongsTo=parr
426	although only a very limited range of lipid compositions were, otherFeat=[]-->, belongsTo=parr
427	tested in this study., otherFeat=[]-->, belongsTo=parr
428	Electron crystallography of AQP0 2D crystals makes it, otherFeat=[]-->, belongsTo=parr
429	possible to visualize the lipids surrounding the membrane, otherFeat=[]-->, belongsTo=parr
430	protein. In our studies, only a single layer of lipid molecules, otherFeat=[]-->, belongsTo=parr
431	separates the individual tetramers in the 2D crystals, otherFeat=[]-->, belongsTo=parr
432	(Figure 2A). This tight packing of the lipids in the 2D crystals, otherFeat=[]-->, belongsTo=parr
433	restricts their mobility and makes it possible to see the lipids, otherFeat=[]-->, belongsTo=parr
434	in the density maps, providing us with the opportunity to, otherFeat=[]-->, belongsTo=parr
435	study the interactions of annular lipids with AQP0. However,, otherFeat=[]-->, belongsTo=parr
436	this situation is not typical for a biological membrane, in, otherFeat=[]-->, belongsTo=parr
437	which membrane proteins tend to be separated by a larger, otherFeat=[]-->, belongsTo=parr
438	number of lipids. Nevertheless, the 2D crystals formed, otherFeat=[]-->, belongsTo=parr
439	in vitro display the same lattice parameters as the 2D arrays, otherFeat=[]-->, belongsTo=parr
440	formed by AQP0 in native lens membranes (Buzhynskyy, otherFeat=[]-->, belongsTo=parr
441	et al, 2007). The 2D crystals are therefore a good representa-, otherFeat=[]-->, belongsTo=parr
442	tion of the AQP0 arrays in the lens membrane. Furthermore,, otherFeat=[]-->, belongsTo=parr
443	AQP0 forms 2D crystals with lipids as different as DMPC and, otherFeat=[]-->, belongsTo=parr
444	EPLs. The sandwiching between two adjacent tetramers may, otherFeat=[]-->, belongsTo=parr
445	thus limit the mobility of the lipids but may not impose, otherFeat=[]-->, belongsTo=parr
446	further restrictions on the behaviour of the lipids surrounding, otherFeat=[]-->, belongsTo=parr
447	AQP0. Our ?ndings concerning the interaction of annular, otherFeat=[]-->, belongsTo=parr
448	lipids with AQP0 in the 2D crystals should therefore bear, otherFeat=[]-->, belongsTo=parr
449	relevance for membrane proteins surrounded by a larger, otherFeat=[]-->, belongsTo=parr
450	number of lipids. Nevertheless, further studies will be, otherFeat=[]-->, belongsTo=parr
451	required to con?rm this notion., otherFeat=[]-->, belongsTo=parr
452	The structures of AQP0 in two different lipid environments, otherFeat=[]-->, belongsTo=parr
453	allow us to see how the protein and the lipid bilayer adapt to, otherFeat=[]-->, belongsTo=parr
454	each other. To accommodate proteins in a lipid bilayer with a, otherFeat=[]-->, belongsTo=parr
455	different hydrophobic thickness, a situation known as hydro-, otherFeat=[]-->, belongsTo=parr
456	phobic mismatch, it has been proposed that either the lipids, otherFeat=[]-->, belongsTo=parr
457	Figure 4 EPL headgroups and acyl chains. (A) Lipid PE3 interacts, otherFeat=[]-->, belongsTo=fig_caption
458	with AQP0 through a putative lipid-binding motif formed by Arg196, otherFeat=[]-->, belongsTo=fig_caption
459	and Tyr105. (B) In AQP0 DMPC the same residues do not interact with, otherFeat=[]-->, belongsTo=fig_caption
460	the corresponding lipid, PC3. (C) Lipid PE7 in AQP0EPL bends to, otherFeat=[]-->, belongsTo=fig_caption
461	follow the surface features of AQP0 and ?lls in a pocket formed by, otherFeat=[]-->, belongsTo=fig_caption
462	residues Trp10, Val91, and Leu95. (D) The corresponding lipid in, otherFeat=[]-->, belongsTo=fig_caption
463	AQP0DMPC, PC7, follows a different path but ?lls in the same pocket, otherFeat=[]-->, belongsTo=fig_caption
464	in the AQP0 surface., otherFeat=[]-->, belongsTo=fig_caption
465	Interaction of AQPO with E. coli lipids, otherFeat=[]-->, belongsTo=nota_cab_pie
466	RK Hite et al, otherFeat=[]-->, belongsTo=nota_cab_pie
467	The EMBO Journal, otherFeat=[]-->, belongsTo=nota_cab_pie
468	VOL 29, otherFeat=['U']-->, belongsTo=nota_cab_pie
469	|, otherFeat=[]-->, belongsTo=nota_cab_pie
470	NO 10, otherFeat=['U']-->, belongsTo=nota_cab_pie
471	|, otherFeat=[]-->, belongsTo=nota_cab_pie
472	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
473	& 2010 European Molecular Biology Organization, otherFeat=[]-->, belongsTo=nota_cab_pie
474	1656, otherFeat=[u'b']-->, belongsTo=nota_cab_pie
475	adjust their length to match the protein or that a-helical, otherFeat=[]-->, belongsTo=parr
476	membrane proteins may stretch or contract axially to adapt, otherFeat=[]-->, belongsTo=parr
477	to the thickness of the surrounding lipid bilayer (Lee, 2004)., otherFeat=[]-->, belongsTo=parr
478	It has been shown for several membrane proteins that, otherFeat=[]-->, belongsTo=parr
479	hydrophobic mismatch affects their activity (Lee, 2004),, otherFeat=[]-->, belongsTo=parr
480	suggesting changes in protein structure. It is not clear, how-, otherFeat=[]-->, belongsTo=parr
481	ever, whether all membrane proteins have the ?exibility to, otherFeat=[]-->, belongsTo=parr
482	adjust to the hydrophobic thickness of the surrounding lipid, otherFeat=[]-->, belongsTo=parr
483	bilayer. Aquaporin-0, which is a very stable and presumably a, otherFeat=[]-->, belongsTo=parr
484	very rigid membrane protein, has the same structure in the, otherFeat=[]-->, belongsTo=parr
485	EPL and DMPC bilayers (Figure 1D). In the two structures, it, otherFeat=[]-->, belongsTo=parr
486	is exclusively the lipids that adapt to the protein, which bend, otherFeat=[]-->, belongsTo=parr
487	and interdigitate to accommodate the longer acyl chains of, otherFeat=[]-->, belongsTo=parr
488	the EPLs. A caveat of this observation is that the acyl chains, otherFeat=[]-->, belongsTo=parr
489	of EPLs are not only longer than those of DMPC, but 55% of, otherFeat=[]-->, belongsTo=parr
490	the acyl chains of EPLs are unsaturated (Lugtenberg and, otherFeat=[]-->, belongsTo=parr
491	Peters, 1976), and double bonds reduce the hydrophobic, otherFeat=[]-->, belongsTo=parr
492	thickness of the bilayer formed by unsaturated lipids, otherFeat=[]-->, belongsTo=parr
493	(Marsh, 2008). Thus, although the AQP0DMPC and AQP0EPL, otherFeat=[]-->, belongsTo=parr
494	structures suggest that it is exclusively the lipids that adapt to, otherFeat=[]-->, belongsTo=parr
495	the protein, the hydrophobic thickness of the DMPC and EPL, otherFeat=[]-->, belongsTo=parr
496	bilayers may be too similar to induce observable changes in, otherFeat=[]-->, belongsTo=parr
497	the AQP0 structure. To conclusively rule out structural changes, otherFeat=[]-->, belongsTo=parr
498	in AQP0 due to hydrophobic mismatch, it will be necessary to, otherFeat=[]-->, belongsTo=parr
499	determine structures of AQP0, in which the protein is sur-, otherFeat=[]-->, belongsTo=parr
500	rounded by lipids that form bilayers with a hydrophobic, otherFeat=[]-->, belongsTo=parr
501	thickness that is signi?cantly different from that of AQP0., otherFeat=[]-->, belongsTo=parr
502	After modelling all the headgroups as ethanolamine and, otherFeat=[]-->, belongsTo=parr
503	re?ning the lipids in the AQP0EPL structure, we did not, otherFeat=[]-->, belongsTo=parr
504	observe any density that indicated PG or CL headgroups., otherFeat=[]-->, belongsTo=parr
505	This result suggests that there are no lipid positions on the, otherFeat=[]-->, belongsTo=parr
506	surface of AQP0 that are preferentially occupied by PG or CL, otherFeat=[]-->, belongsTo=parr
507	lipids. In the absence of preferential binding sites, averaging, otherFeat=[]-->, belongsTo=parr
508	of the three headgroups would probably result in all, otherFeat=[]-->, belongsTo=parr
509	headgroups appearing as the predominant PE headgroup., otherFeat=[]-->, belongsTo=parr
510	Furthermore, inspection of the protein surface did not reveal, otherFeat=[]-->, belongsTo=parr
511	speci?c CL-binding sites as de?ned by Palsdottir and Hunte, otherFeat=[]-->, belongsTo=parr
512	(2004). As CL, a lipid composed of four acyl chains and two, otherFeat=[]-->, belongsTo=parr
513	phosphodiester groups linked by a glycerol, constitutes only, otherFeat=[]-->, belongsTo=parr
514	10% of EPLs, it may randomly occupy two adjacent lipid, otherFeat=[]-->, belongsTo=parr
515	positions and thus be obscured as a result of averaging., otherFeat=[]-->, belongsTo=parr
516	Alternatively, due to its larger size, CL may preferentially, otherFeat=[]-->, belongsTo=parr
517	localize to the larger lipid area in the middle of four neigh-, otherFeat=[]-->, belongsTo=parr
518	bouring AQP0 tetramers in which no ordered lipid molecules, otherFeat=[]-->, belongsTo=parr
519	were found (asterisks in Figure 2A). As PG and CL lipids do, otherFeat=[]-->, belongsTo=parr
520	not occur in lens membranes, it is not surprising that AQP0, otherFeat=[]-->, belongsTo=parr
521	does not select for these lipids at particular positions., otherFeat=[]-->, belongsTo=parr
522	Despite the different acyl chain length and the different, otherFeat=[]-->, belongsTo=parr
523	orientations of the headgroups, the average distance between, otherFeat=[]-->, belongsTo=parr
524	the phosphodiester groups in the two lea?ets is almost, otherFeat=[]-->, belongsTo=parr
525	identical in the bilayers formed by EPLs, 31.9 A? , and DMPC,, otherFeat=[]-->, belongsTo=parr
526	33.6 A? (Figure 3A). More surprisingly, the average distance, otherFeat=[]-->, belongsTo=parr
527	between the C2 atoms of the glycerol of the lipids in the two, otherFeat=[]-->, belongsTo=parr
528	lea?ets is larger for the shorter DMPC lipids, 31.2 A? , than for, otherFeat=[]-->, belongsTo=parr
529	the longer EPLs, 27.0 A? (Figure 3B). This ?nding suggests that, otherFeat=[]-->, belongsTo=parr
530	the vertical position of the lipids is de?ned by the strong, otherFeat=[]-->, belongsTo=parr
531	charges of the phosphodiester groups that have to be posi-, otherFeat=[]-->, belongsTo=parr
532	tioned outside the hydrophobic belt of the membrane protein., otherFeat=[]-->, belongsTo=parr
533	The glycerol backbone, however, which is largely hydropho-, otherFeat=[]-->, belongsTo=parr
534	bic in nature, is allowed more ?exibility in its positioning, otherFeat=[]-->, belongsTo=parr
535	within the hydrophobic region of the membrane protein and, otherFeat=[]-->, belongsTo=parr
536	can occupy the same region as the acyl chains., otherFeat=[]-->, belongsTo=parr
537	In conclusion, we propose that the vertical position of the, otherFeat=[]-->, belongsTo=parr
538	annular lipids surrounding a membrane protein is de?ned by, otherFeat=[]-->, belongsTo=parr
539	the thickness of the hydrophobic surface of the membrane, otherFeat=[]-->, belongsTo=parr
540	protein, with the charged phosphodiester groups of the lipids, otherFeat=[]-->, belongsTo=parr
541	in the two lea?ets being positioned immediately outside of, otherFeat=[]-->, belongsTo=parr
542	the hydrophobic belt of the protein. Although some mem-, otherFeat=[]-->, belongsTo=parr
543	brane proteins may change their structure to adjust to, otherFeat=[]-->, belongsTo=parr
544	bilayers with a signi?cantly different hydrophobic thickness,, otherFeat=[]-->, belongsTo=parr
545	it remains to be determined whether rigid membrane pro-, otherFeat=[]-->, belongsTo=parr
546	teins, such as AQP0, can adapt in a similar manner. The, otherFeat=[]-->, belongsTo=parr
547	requirement that acyl chains ?ll in the gaps on the protein, otherFeat=[]-->, belongsTo=parr
548	surface constitutes the guiding principle for the lateral inter-, otherFeat=[]-->, belongsTo=parr
549	actions of annular lipids with membrane proteins. Additional, otherFeat=[]-->, belongsTo=parr
550	interactions of the protein with headgroups of annular lipids,, otherFeat=[]-->, belongsTo=parr
551	that is, lipid-binding sites, may allow membrane proteins to, otherFeat=[]-->, belongsTo=parr
552	select for speci?c lipids and/or to form tight interactions with, otherFeat=[]-->, belongsTo=parr
553	lipids important for their function or structural integrity., otherFeat=[]-->, belongsTo=parr
554	Furthermore, the fact that AQP0 forms well-ordered 2D, otherFeat=[]-->, belongsTo=parr
555	crystals with lipids as different as DMPC and EPLs suggests, otherFeat=[]-->, belongsTo=parr
556	that it will be possible to grow 2D crystals of AQP0 with many, otherFeat=[]-->, belongsTo=parr
557	other lipids, opening an avenue for the systematic study of, otherFeat=[]-->, belongsTo=parr
558	lipid-protein interactions., otherFeat=[]-->, belongsTo=parr
559	Materials and methods, otherFeat=[u'b']-->, belongsTo=title
560	Protein puri?cation and crystallization, otherFeat=[u'b']-->, belongsTo=parrnote
561	The core tissue of sheep lenses (Wolverine Packing Company,, otherFeat=[]-->, belongsTo=parrnote
562	Detroit, MI) was dissected away from the soft cortical tissue and, otherFeat=[]-->, belongsTo=parrnote
563	membranes were prepared as previously described (Gonen et al,, otherFeat=[]-->, belongsTo=parrnote
564	2004). Puri?ed membranes were solubilized in 4% (w/v) octyl, otherFeat=[]-->, belongsTo=parrnote
565	glucoside in 10 mM Tris (pH 8.0) for 30 min at 221C. Insoluble, otherFeat=[]-->, belongsTo=parrnote
566	material was separated by centrifugation at 300 000 ? g. Solubilized, otherFeat=[]-->, belongsTo=parrnote
567	proteins were bound to a MonoQ column (GE Healthcare) and, otherFeat=[]-->, belongsTo=parrnote
568	eluted with 150 mM NaCl. Pooled fractions were run over a, otherFeat=[]-->, belongsTo=parrnote
569	Superose 12 (GE Healthcare) and eluted with 1.2% octyl glucoside, otherFeat=[]-->, belongsTo=parrnote
570	in 10 mM Tris (pH 8.0) and 150 mM NaCl. Puri?ed AQP0 was, otherFeat=[]-->, belongsTo=parrnote
571	reconstituted into 2D crystals using E. coli polar lipids (Avanti Polar, otherFeat=[]-->, belongsTo=parrnote
572	Lipids) at a lipid-to-protein ratio of 0.25 (mg/mg) by dialysis against, otherFeat=[]-->, belongsTo=parrnote
573	10 mM MES (pH 6), 100 mM NaCl, and 50 mM MgCl2 at 371C for, otherFeat=[]-->, belongsTo=parrnote
574	1 week with daily buffer exchanges., otherFeat=[]-->, belongsTo=parrnote
575	Data collection, data processing, model building,, otherFeat=[u'b']-->, belongsTo=parrnote
576	and re?nement, otherFeat=[u'b']-->, belongsTo=parrnote
577	Specimens for cryo-electron microscopy were prepared using the, otherFeat=[]-->, belongsTo=parrnote
578	carbon sandwich technique (Gyobu et al, 2004). Grids were, otherFeat=[]-->, belongsTo=parrnote
579	transferred into an FEI Tecnai Polara electron microscope operated, otherFeat=[]-->, belongsTo=parrnote
580	at an acceleration voltage of 300 kV. Low-dose electron diffraction, otherFeat=[]-->, belongsTo=parrnote
581	patterns were recorded at liquid helium temperature (B6 K) with a, otherFeat=[]-->, belongsTo=parrnote
582	4 ? 4 K CCD camera (Gatan) and a camera length of 1.9 m. Electron, otherFeat=[]-->, belongsTo=parrnote
583	diffraction patterns were analysed and merged as described, otherFeat=[]-->, belongsTo=parrnote
584	previously (Gonen et al, 2004). The structure of AQP0 was, otherFeat=[]-->, belongsTo=parrnote
585	determined by molecular replacement in Phaser 2.1 (McCoy et al,, otherFeat=[]-->, belongsTo=parrnote
586	2007) using as search model the 1.9-A? electron crystallographic, otherFeat=[]-->, belongsTo=parrnote
587	model of AQP0 without the C-terminal helix ((Gonen et al, 2005);, otherFeat=[]-->, belongsTo=parrnote
588	PDB ID: 2B6O)., otherFeat=['U']-->, belongsTo=parrnote
589	The protein was rebuilt in Coot (Emsley and Cowtan, 2004),, otherFeat=[]-->, belongsTo=parrnote
590	and the model was re?ned in CNS version 1.2 (Brunger et al, 1998), otherFeat=[]-->, belongsTo=parrnote
591	using 300 kV electron scattering factors. Protein residues 7?226, otherFeat=[]-->, belongsTo=parrnote
592	were visible and were modelled and re?ned., otherFeat=[]-->, belongsTo=parrnote
593	Building the lipids into the density map was complicated by the, otherFeat=[]-->, belongsTo=parrnote
594	structural heterogeneity of EPLs. All lipids were initially modelled, otherFeat=[]-->, belongsTo=parrnote
595	into the Fo?F c difference map with PE headgroups and short (B8?10, otherFeat=[]-->, belongsTo=parrnote
596	carbon) acyl chains. Short acyl chains were used because some of, otherFeat=[]-->, belongsTo=parrnote
597	the chains were poorly de?ned in the initial difference map and, otherFeat=[]-->, belongsTo=parrnote
598	because several of the densities representing acyl chains were, otherFeat=[]-->, belongsTo=parrnote
599	branched. After a round of simulated annealing re?nement, further, otherFeat=[]-->, belongsTo=parrnote
600	carbons were added to the acyl chains if additional density was, otherFeat=[]-->, belongsTo=parrnote
601	clearly visible in the composite omit map. For branched densities,, otherFeat=[]-->, belongsTo=parrnote
602	the strongest arm was chosen for model building. This process was, otherFeat=[]-->, belongsTo=parrnote
603	repeated iteratively until up to a maximum of 18 carbon atoms were, otherFeat=[]-->, belongsTo=parrnote
604	Interaction of AQPO with E. coli lipids, otherFeat=[]-->, belongsTo=nota_cab_pie
605	RK Hite et al, otherFeat=[]-->, belongsTo=nota_cab_pie
606	& 2010 European Molecular Biology Organization, otherFeat=[]-->, belongsTo=nota_cab_pie
607	The EMBO Journal, otherFeat=[]-->, belongsTo=nota_cab_pie
608	VOL 29, otherFeat=['U']-->, belongsTo=nota_cab_pie
609	|, otherFeat=[]-->, belongsTo=nota_cab_pie
610	NO 10, otherFeat=['U']-->, belongsTo=nota_cab_pie
611	|, otherFeat=[]-->, belongsTo=nota_cab_pie
612	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
613	1657, otherFeat=[u'b']-->, belongsTo=nota_cab_pie
614	built (the longest abundant acyl chain length in EPLs) or until no, otherFeat=[]-->, belongsTo=parrnote
615	additional density appeared upon further cycles. Figures were, otherFeat=[]-->, belongsTo=parrnote
616	prepared with PyMol (www.pymol.org)., otherFeat=[]-->, belongsTo=parrnote
617	Accession codes, otherFeat=[u'b']-->, belongsTo=parrnote
618	Atomic coordinates and structure factor ?les have been deposited, otherFeat=[]-->, belongsTo=parrnote
619	with the Protein Data Bank under the accession code 3M9I., otherFeat=[]-->, belongsTo=parrnote
620	Supplementary data, otherFeat=[u'b']-->, belongsTo=parrnote
621	Supplementary data are available at The EMBO Journal Online, otherFeat=[]-->, belongsTo=parrnote
622	(http://www.embojournal.org)., otherFeat=[u'a']-->, belongsTo=parrnote
623	Acknowledgements, otherFeat=[u'b']-->, belongsTo=title
624	This study was supported by NIH grant R01 EY015107 (to TW). TW, otherFeat=[]-->, belongsTo=parrnote
625	is an investigator of the Howard Hughes Medical Institute. We thank, otherFeat=[]-->, belongsTo=parrnote
626	S Harrison and Y Fujiyoshi for continuous support and advice;, otherFeat=[]-->, belongsTo=parrnote
627	K Abe for advice regarding specimen preparation and T Rapoport, otherFeat=[]-->, belongsTo=parrnote
628	for insightful discussions. RKH and TW conceived and designed, otherFeat=[]-->, belongsTo=parrnote
629	the project. RKH performed the experiments. ZL assisted with data, otherFeat=[]-->, belongsTo=parrnote
630	collection. RKH and TW wrote the paper., otherFeat=[]-->, belongsTo=parrnote
631	Con?ict of interest, otherFeat=[u'b']-->, belongsTo=title
632	The authors declare that they have no con?ict of interest., otherFeat=[]-->, belongsTo=parrnote
633	References, otherFeat=[u'b']-->, belongsTo=title
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697	The EMBO Journal is published by Nature, otherFeat=[]-->, belongsTo=parr
698	Publishing Group on behalf of European, otherFeat=[]-->, belongsTo=parr
699	Molecular Biology Organization. This article is licensed, otherFeat=[]-->, belongsTo=parr
700	under a Creative Commons Attribution-Noncommercial-, otherFeat=[]-->, belongsTo=parr
701	Share Alike 3.0 Licence. [http://creativecommons.org/, otherFeat=[]-->, belongsTo=parr
702	licenses/by-nc-sa/3.0/], otherFeat=[]-->, belongsTo=parr
703	Interaction of AQPO with E. coli lipids, otherFeat=[]-->, belongsTo=nota_cab_pie
704	RK Hite et al, otherFeat=[]-->, belongsTo=nota_cab_pie
705	The EMBO Journal, otherFeat=[]-->, belongsTo=nota_cab_pie
706	VOL 29, otherFeat=['U']-->, belongsTo=nota_cab_pie
707	|, otherFeat=[]-->, belongsTo=nota_cab_pie
708	NO 10, otherFeat=['U']-->, belongsTo=nota_cab_pie
709	|, otherFeat=[]-->, belongsTo=nota_cab_pie
710	2010, otherFeat=[]-->, belongsTo=nota_cab_pie
711	& 2010 European Molecular Biology Organization, otherFeat=[]-->, belongsTo=nota_cab_pie
712	1658, otherFeat=[u'b']-->, belongsTo=nota_cab_pie
==============================
0	EMBOopen-->id=0, page=0, size=9, fam=Times, col=#231f20, type=title, textLines=12--->[]--->note
1	Principles of membrane protein interactions with annular lipids deduced from aquaporin-0 2D crystals-->id=1, page=0, size=13, fam=Times, col=#000000, type=title, textLines=3--->[u'b']--->title
2	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.-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
3	Richard K Hite1, Zongli Li1,2 and Thomas Walz1,2,*-->id=0, page=0, size=9, fam=Times, col=#231f20, type=title, textLines=12--->[u'b']--->title
4	1Department of Cell Biology, Harvard Medical School, Boston, MA, USA and 2Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
5	We have previously described the interactions of aquaporin-0 (AQP0) with dimyristoyl phosphatidylcholine (DMPC) lipids. We have now determined the 2.5 A˚ structure 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 annular 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 responsible 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.-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
6	The EMBO Journal (2010) 29, 1652–1658. doi:10.1038/ emboj.2010.68; Published online 13 April 2010-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
7	Subject Categories: membranes & transport; structural biology-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
8	Keywords: electron crystallography; lens; lipid–protein interactions; water channel-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
9	Introduction-->id=0, page=0, size=9, fam=Times, col=#231f20, type=title, textLines=12--->[u'b']--->title
10	How do membrane proteins interact with lipids? Spin-labelling and fluorescence-quenching studies have provided a thermodynamic understanding of lipid–protein 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 lipid–protein interactions comes from lipids in crystal structures 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 established 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-crystallized lipids originate from the donor membrane and must have remained associated with the protein during solubilization and purification to be incorporated in the three-dimensional (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 annular lipids form only weak and non-specific interactions with membrane proteins (Lee, 2003). Generic annular lipids are thus typically lost during solubilization and/or purification of membrane proteins and are usually not observed in crystal structures.-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
11	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.-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
12	Previously, the structure of the lens-specific water channel aquaporin-0 (AQP0) was determined by electron crystallography 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 electron 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 in between two AQP0 tetramers, which is not typically the case for lipids surrounding membrane proteins in biological membranes.-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
13	Received: 7 January 2010; accepted: 23 March 2010; published online: 13 April 2010-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
14	*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-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
15	The EMBO Journal (2010) 29, 1652–1658 | & 2010 European Molecular Biology Organization | Some Rights Reserved 0261-4189/10-->id=7, page=0, size=4, fam=Times, col=#000000, type=parrnote, textLines=16--->[]--->note
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28	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 lipid–protein interactions. If AQP0 2D crystals could be grown with other lipids, such crystals would provide an opportunity to study how a membrane 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), phosphatidylglycerol (PG) and cardiolipin (CL) rather than the pure phosphatidylcholine (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.-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
29	Results-->id=0, page=0, size=9, fam=Times, col=#231f20, type=title, textLines=12--->[u'b']--->title
30	Structure of AQP0 in DMPC and EPL bilayers-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[u'b']--->title
31	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 diffraction 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 7–226) in the membrane junction (Figure 1C; Table I). Residues 1–6 and 227–263, 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.-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
32	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-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
33	(Figure 1E).-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[u'a']--->parr
34	Modelling the EPL bilayer-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[u'b']--->title
35	In addition to the protein, the density map also allowed us to build all seven annular lipids surrounding each AQP0 monomer, 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 conformations. 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-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
36	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 2Fo –Fc 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 2Fo–Fc density map for the water molecules is shown at a contouring level of 1s (blue wire mesh).-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->capfig
37	Interaction of AQPO with E. coli lipids-->id=7, page=0, size=4, fam=Times, col=#000000, type=parrnote, textLines=16--->[]--->note
38	RK Hite et al-->id=7, page=0, size=4, fam=Times, col=#000000, type=parrnote, textLines=16--->[]--->note
39	& 2010 European Molecular Biology Organization-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->note
40	The EMBO Journal-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->note
41	VOL 29-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->['U']--->note
42	|-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->note
43	NO 10-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->['U']--->note
44	|-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->note
45	2010-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->note
46	1653-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[u'b']--->note
47	branches disappeared, and all acyl chains were represented by a single, mostly continuous density.-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
48	Approximately 55% of the acyl chains of EPLs contain an unsaturated bond, with the two most abundant species being 16c1:9 and 18c1:11 (Lugtenberg and Peters, 1976). Although some lipids showed kinks that could indicate the presence of a double bond, due to the heterogeneity of the acyl chains in EPLs, we modelled all acyl chains as being fully saturated.-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
49	Escherichia coli polar lipids are a mixture of three different headgroups (67% PE, 23% PG and 10% CL lipids; Oursel et al (2007)) with PE being by far the most abundant headgroup. We therefore initially modelled all headgroups as PE. After refinement, we found no evidence in the density map, which suggested that any of the lipid positions is preferentially occupied by a lipid with a particular headgroup. Our final model of the EPL bilayer thus contains seven PE molecules with saturated acyl chains ranging from 5 to 17 carbon atoms-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
50	(Figure 2B).-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[u'a']--->parr
51	Organization of the EPL and DMPC bilayers surrounding AQP0-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[u'b']--->title
52	The EPL and DMPC bilayers surrounding AQP0 are remark--->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
53	ably similar (Figure 2B and C). The two bilayers contain the same number of lipids at comparable positions (Figure 3C) and have almost the same thickness (the average distance between the phosphodiester groups in the two leaflets is 31.9 A˚ for the EPL bilayer and 33.6 A˚ for the DMPC bilayer; Figure 3A). As acyl chains of EPLs are on average longer than those of DMPC (16 versus 14 carbon atoms), this finding raises the question how the longer EPL acyl chains are accommodated. Unexpectedly, despite the longer acyl chains of EPLs, the average distance between the C2 atoms of glycerols in the two leaflets of the EPL bilayer, 27.0 A˚ , is smaller than the corresponding average distance in the DMPC bilayer, 31.2 A˚ (Figure 3B). Further comparison of the AQP0 EPL and AQP0DMPC structures reveals that the DMPC molecules cover less surface area on AQP0 than EPLs (Figures 2B and C). Indeed, the DMPC bilayer leaves areas of the hydrophobic surface of AQP0 uncovered (Figure 2C, e.g. area in between PC3 and PC4 of the extracellular leaflet and-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
54	Figure 2 The EPL bilayer. (A) Top view of the AQP0 2D crystal showing AQP0 tetramers (gold) and the surrounding EPLs (red). (B, C) The seven (B) annular EPLs and (C) DMPC molecules surrounding an AQP0 monomer. As lipids are sandwiched between two adjacent AQP0 subunits, their positions relative to both AQP0 subunits are shown.-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->capfig
55	Table I Electron crystallographic data-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
56	Two-dimensional crystals Layer group p422 Unit cell a ¼ b ¼ 65.5 A˚ Thickness (assumed) 200 A˚-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
57	Electron diffraction-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
58	Number of patterns merged 281 (01: 11; 201: 22;-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
59	451: 63; 601: 108; 701: 77) Resolution limit for merging 2.3 A˚-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
60	RFriedel 18.9% RMerge 22.6% Observed amplitudes to 2.5 A˚ 129 893 Unique reflections 14 417 Maximum tilt angle 74.21-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
61	Fourier space sampled 92.3% (83.5% at 2.6–2.5 A˚ ) Multiplicity 8.1 (4.0 at 2.6–2.5 A˚ )-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
62	Crystallographic refinement (10.0–2.5 A˚)-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
63	Resolution limit for refinement 2.5 A˚-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
64	Crystallographic R factor 24.98% Free R factor 28.43%-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
65	Reflections in working/test set 12 801/1453 Non-hydrogen protein atoms 1668-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
66	Non-hydrogen lipid atoms 273 Solvent molecules 8-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
67	Average protein B factor (A˚ 2) 42.3 Average lipid B factor (A˚ 2) 88.0-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
68	Ramachandran plot (%) 98.4/1.6/0.0 (allowed;-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
69	generous; disallowed)-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
70	Rfree is calculated from a randomly chosen 10% of reflections, and Rcryst is calculated over the remaining 90% of reflections.-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
71	Interaction of AQPO with E. coli lipids-->id=7, page=0, size=4, fam=Times, col=#000000, type=parrnote, textLines=16--->[]--->note
72	RK Hite et al-->id=7, page=0, size=4, fam=Times, col=#000000, type=parrnote, textLines=16--->[]--->note
73	The EMBO Journal-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->note
74	VOL 29-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->['U']--->note
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76	NO 10-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->['U']--->note
77	|-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->note
78	2010-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->note
79	& 2010 European Molecular Biology Organization-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->note
80	1654-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[u'b']--->note
81	PC7 of the cytoplasmic leaflet), suggesting that 14-carbon acyl chains are close to the minimum needed to saturate the hydrophobic belt of AQP0. Furthermore, unlike the lipids in the DMPC bilayer, several of the longer EPL acyl chains interdigitate in the middle of the bilayer, filling gaps between acyl chains in the opposite leaflet (Figure 2B; e.g. one acyl chain of PE7 inserts between the two acyl chains of PE3 and the other acyl chain of PE7 inserts between the two acyl chains of PE4). Other acyl chains bend sharply as they approach the midpoint of the bilayer and then extend parallel to the membrane plane (Figure 2B; e.g. acyl chains of PE4, PE6, and PE7). Such bending may be facilitated by unsaturated bonds present in acyl chains of EPLs, which have previously been proposed to explain the bent conformations seen in lipids associated with cytochrome b–c1 complexes (Palsdottir and Hunte, 2004). Indeed, the positions of the kinks seen in the acyl chains of lipids PE3, PE4, and PE6 occur close to the positions of the double bonds in the most abundant unsaturated acyl chains of EPLs, 16c1:9 and 18c1:11, whereas the kink in the acyl chain of PE7 is located between C6 and C7.-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
82	Interaction of the lipid headgroups with AQP0-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[u'b']--->title
83	The AQP0EPL and AQP0DMPC structures make it possible to analyse the interactions of AQP0 with different lipids. Although the corresponding lipids in the two bilayers are at very similar positions (Figure 3C), all the headgroups of the corresponding lipids in the two bilayers adopt different conformations. Furthermore, none of the DMPC lipids in the AQP0DMPC structure interacted with AQP0 through a lipidbinding motif (Gonen et al, 2005), and only one lipid, PE3, in the AQP0EPL structure is in an environment consistent with a lipid-binding motif: the phosphodiester group of PE3 interacts with a positively charged residue, Arg 196, and a polar residue, Tyr 105 (Figure 4A). In PC3, the corresponding lipid in the AQP0DMPC structure (Figure 4B), the glycerol backbone is shifted by B3.4 A˚ closer to the centre of the lipid bilayer. When Hunte and coworkers identified the lipid-binding motif, they noted that it did not seem to apply to PC lipids and suggested that this may be due to the large choline headgroup that could cause steric clashes (Palsdottir and Hunte, 2004). We were therefore unsure whether the changes in the conformation of PE3 with respect to PC3 were the result of the presence of a lipid-binding motif. When we attempted to reposition PC3 to a location more similar to PE3, we found that there was sufficient space for a choline headgroup to occupy the same space as the ethanolamine headgroup of PE3. As the choline headgroup of PC3 does not occupy this position, even though it would be sterically possible, and therefore does not interact with AQP0 through Arg 196 and Tyr 105, we conclude that these two residues do not constitute a true lipid-binding motif. Together, these observations suggest that the exact chemical identities of the phospholipid headgroups have a negligible role in the interaction of annular lipids with membrane proteins, corroborating previous results obtained by spin-labelling studies (Lee, 2003).-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
84	Figure 3 Comparisons between the EPL and DMPC bilayers. (A) The average distance between the phosphodiester groups in the two leaflets of the bilayer formed by EPLs (31.9 A˚ ) is very similar to that between the phosphodiester groups in the DMPC bilayers (33.6 A˚). Phosphorous atoms are shown in orange. Red shading represents the region between the most distantly located phosphorous atoms in each leaflet. (B) Despite the longer acyl chains of EPLs, the average distance between the C2 atoms of the lipids’ glycerol backbones in the two leaflets of the bilayer formed by EPLs (27.0 A˚ ) is shorter than that between the C2 atoms in the DMPC bilayers (31.2 A˚). The glycerol groups are shown in green. The green shading represents the region between the two most distantly located C2 atoms in each leaflet. (C) Overlay of the lipids seen in the AQP0EPL (red) and AQP0DMPC (blue) structures. (D) Same as in (C) with the lipids coloured according to their B-factors. Colour coding: intense blue, B-factors below 75 A˚ 2; pale blue, B-factors between 75 and 94 A˚ 2 ; pale red, B-factors between 95 and 114 A˚ 2; intense red, B-factors above 115 A˚ 2.-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->capfig
85	Interaction of AQPO with E. coli lipids-->id=7, page=0, size=4, fam=Times, col=#000000, type=parrnote, textLines=16--->[]--->note
86	RK Hite et al-->id=7, page=0, size=4, fam=Times, col=#000000, type=parrnote, textLines=16--->[]--->note
87	& 2010 European Molecular Biology Organization-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->note
88	The EMBO Journal-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->note
89	VOL 29-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->['U']--->note
90	|-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->note
91	NO 10-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->['U']--->note
92	|-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->note
93	2010-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->note
94	1655-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[u'b']--->note
95	Interaction of the lipid acyl chains with AQP0-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[u'b']--->title
96	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).-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
97	The positions of the acyl chains in the cytoplasmic leaflet vary to a greater degree between AQP0 EPL 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 mobility is more restricted compared with those in the cytoplasmic leaflet (Figure 3D and Supplementary Figure S3).-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
98	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 3 A˚ 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 corresponding 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.-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
99	Discussion-->id=0, page=0, size=9, fam=Times, col=#231f20, type=title, textLines=12--->[u'b']--->title
100	Comparison of our new AQP0EPL structure with the previously determined AQP0DMPC structure shows that the annular 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.-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
101	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 representation 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.-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
102	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 hydrophobic mismatch, it has been proposed that either the lipids 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 hydrophobic mismatch affects their activity (Lee, 2004), suggesting changes in protein structure. It is not clear, however, 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 the acyl chains of EPLs are unsaturated (Lugtenberg and Peters, 1976), and double bonds reduce the hydrophobic thickness of the bilayer formed by unsaturated lipids (Marsh, 2008). Thus, although the AQP0DMPC and AQP0EPL 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 surrounded by lipids that form bilayers with a hydrophobic thickness that is significantly different from that of AQP0.-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
103	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 AQP0 DMPC 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.-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->capfig
104	Interaction of AQPO with E. coli lipids-->id=7, page=0, size=4, fam=Times, col=#000000, type=parrnote, textLines=16--->[]--->note
105	RK Hite et al-->id=7, page=0, size=4, fam=Times, col=#000000, type=parrnote, textLines=16--->[]--->note
106	The EMBO Journal-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->note
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111	2010-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->note
112	& 2010 European Molecular Biology Organization-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->note
113	1656-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[u'b']--->note
114	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 specific CL-binding sites as defined by Palsdottir and Hunte (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 neighbouring 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.-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
115	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˚ (Figure 3A). More surprisingly, the average distance 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˚ (Figure 3B). This finding suggests that the vertical position of the lipids is defined by the strong charges of the phosphodiester groups that have to be positioned outside the hydrophobic belt of the membrane protein. The glycerol backbone, however, which is largely hydrophobic 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 membrane proteins may change their structure to adjust to bilayers with a significantly different hydrophobic thickness, it remains to be determined whether rigid membrane proteins, 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 interactions 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.-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
116	Materials and methods-->id=8, page=0, size=8, fam=Times, col=#000000, type=title, textLines=7--->[u'b']--->title
117	Protein purification and crystallization-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[u'b']--->title
118	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.-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
119	Data collection, data processing, model building, and refinement-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[u'b']--->title
120	Specimens for cryo-electron microscopy were prepared using the carbon sandwich technique (Gyobu et al, 2004). Grids were 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 previously (Gonen et al, 2004). The structure of AQP0 was determined by molecular replacement in Phaser 2.1 (McCoy et al, 2007) using as search model the 1.9-A˚ electron crystallographic model of AQP0 without the C-terminal helix ((Gonen et al, 2005);-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
121	PDB ID: 2B6O).-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->['U']--->parr
122	The protein was rebuilt in Coot (Emsley and Cowtan, 2004), and the model was refined in CNS version 1.2 (Brunger et al, 1998) using 300 kV electron scattering factors. Protein residues 7–226 were visible and were modelled and refined.-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
123	Building the lipids into the density map was complicated by the structural heterogeneity of EPLs. All lipids were initially modelled into the Fo–F c difference map with PE headgroups and short (B8–10 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-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
124	Interaction of AQPO with E. coli lipids-->id=7, page=0, size=4, fam=Times, col=#000000, type=parrnote, textLines=16--->[]--->note
125	RK Hite et al-->id=7, page=0, size=4, fam=Times, col=#000000, type=parrnote, textLines=16--->[]--->note
126	& 2010 European Molecular Biology Organization-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->note
127	The EMBO Journal-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->note
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134	built (the longest abundant acyl chain length in EPLs) or until no additional density appeared upon further cycles. Figures were prepared with PyMol (www.pymol.org).-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
135	Accession codes-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[u'b']--->title
136	Atomic coordinates and structure factor files have been deposited with the Protein Data Bank under the accession code 3M9I.-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
137	Supplementary data-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[u'b']--->title
138	Supplementary data are available at The EMBO Journal Online-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
139	(http://www.embojournal.org).-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[u'a']--->parr
140	Acknowledgements-->id=0, page=0, size=9, fam=Times, col=#231f20, type=title, textLines=12--->[u'b']--->title
141	This study was supported by NIH grant R01 EY015107 (to TW). TW is an investigator of the Howard Hughes Medical Institute. We thank S Harrison and Y Fujiyoshi for continuous support and advice; K Abe for advice regarding specimen preparation and T Rapoport for insightful discussions. RKH and TW conceived and designed the project. RKH performed the experiments. ZL assisted with data collection. RKH and TW wrote the paper.-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
142	Conflict of interest-->id=0, page=0, size=9, fam=Times, col=#231f20, type=title, textLines=12--->[u'b']--->title
143	The authors declare that they have no conflict of interest.-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->parr
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178	The EMBO Journal is published by Nature Publishing Group on behalf of European Molecular Biology Organization. This article is licensed under a Creative Commons Attribution-NoncommercialShare Alike 3.0 Licence. [http://creativecommons.org/ licenses/by-nc-sa/3.0/]-->id=2, page=0, size=6, fam=Times, col=#000000, type=parr, textLines=507--->[]--->parr
179	Interaction of AQPO with E. coli lipids-->id=7, page=0, size=4, fam=Times, col=#000000, type=parrnote, textLines=16--->[]--->note
180	RK Hite et al-->id=7, page=0, size=4, fam=Times, col=#000000, type=parrnote, textLines=16--->[]--->note
181	The EMBO Journal-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->note
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186	2010-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->note
187	& 2010 European Molecular Biology Organization-->id=4, page=0, size=5, fam=Times, col=#000000, type=parrnote, textLines=315--->[]--->note
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