Abstract
Aquaporin-0 (AQP0) is a lens-specific water channel that also forms membrane junctions. Reconstitution of AQP0 with dimyristoyl phosphatidylcholine (DMPC) and E. coli polar lipids (EPL) yielded well-ordered, double-layered two-dimensional (2D) crystals that allowed electron crystallographic structure determination of the AQP0-mediated membrane junction. The interacting tetramers in the two crystalline layers are exactly in register, resulting in crystals with p422 symmetry. The high-resolution density maps also allowed modeling of the annular lipids surrounding the tetramers. Comparison of the DMPC and EPL bilayers suggested that the lipid head groups do not play an important role in the interaction of annular lipids with AQP0. We now reconstituted AQP0 with the anionic lipid dimyristoyl phosphatidylglycerol (DMPG), which yielded a mixture of 2D crystals with different symmetries. The different crystal symmetries result from shifts between the two crystalline layers, suggesting that the negatively charged PG head group destabilizes the interaction between the extracellular AQP0 surfaces. Reconstitution of AQP0 with dimyristoyl phosphatidylserine (DMPS), another anionic lipid, yielded crystals that had the usual p422 symmetry, but the crystals showed a pH-dependent tendency to stack through their cytoplasmic surfaces. Finally, AQP0 failed to reconstitute into membranes that were composed of more than 40% dimyristoyl phosphatidic acid (DMPA). Hence, although DMPG, DMPS, and DMPA are all negatively charged lipids, they have very different effects on AQP0 2D crystals, illustrating the importance of the specific lipid head group chemistry beyond its mere charge.
Highlights
The lens-specific water channel aquaporin-0 (AQP0) forms large square arrays in the membranes of fiber cells that allow formation of cell-cell junctions between adjacent cells [1,2].PLOS ONE | DOI:10.1371/journal.pone.0117371 January 30, 2015Effects of Anionic Lipids on AQP0 2D Crystals study design, data collection and analysis, decision to publish, or preparation of the manuscript
The crystals were double-layered, as seen by the two parallel edges of crystals prepared in negative stain (Fig. 1B), and had unit cell dimensions identical to those of AQP0 2D crystals grown with dimyristoyl phosphatidylcholine (DMPC) and E. coli polar lipids (EPL), a = b = 65.5 Å, and γ = 90°, we were not able to merge the diffraction patterns using the established p422 symmetry of double-layered AQP0 2D crystals [4,6]
Comparison of the projection map of the dimyristoyl phosphatidylglycerol (DMPG) crystals with p422 symmetry with those of AQP0 in DMPC or EPL revealed no differences in the projections of AQP0 at a resolution of 7 Å, suggesting that DMPG had not changed the overall structure of the AQP0 tetramer, but rather its packing in the crystals
Summary
The lens-specific water channel aquaporin-0 (AQP0) forms large square arrays in the membranes of fiber cells that allow formation of cell-cell junctions between adjacent cells [1,2]. Reconstitution of purified AQP0 with lipids resulted in double-layered, two-dimensional (2D) crystals that recapitulated the in vivo junctions [3,4] Electron crystallography of these 2D crystals revealed the structure of AQP0 at 1.9 Å resolution, as well as a nearly complete model of the lipid bilayer surrounding the channel; the lipids were modeled as dimyristoyl phosphatidylcholine (DMPC), the synthetic lipid used for 2D crystallization (AQP0DMPC) [5]. Phosphatidylethanolamine (PE), the predominant head group found in EPL, and phosphatidylcholine (PC) are both zwitterionic, which may explain the lack of interactions between AQP0 and the lipid head groups in the structures, as many of the specific lipid—protein interactions identified in biochemical assays or resolved in crystal structures occur between membrane proteins and anionic phospholipids [7]. In the course of these reconstitution experiments, we made the unexpected observation that each of the anionic lipids had a unique effect on the AQP0 2D crystals that formed
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