C OMMUNICATION Co-axial Association of Recombinant Eye Lens Aquaporin-0 Observed in Loosely Packed 3D Crystals Dinesh V. Palanivelu 1 , David E. Kozono 2 , Andreas Engel 1 , Kitaru Suda 1 Ariel Lustig 3 , Peter Agre 2 and Tilman Schirmer 1 * 1 Division of Structural Biology Biozentrum, University of Basel Klingelbergstr. 70, CH-4056 Basel, Switzerland 2 Department of Biological Chemistry and Medicine Johns Hopkins University School of Medicine, Baltimore 21205 MD, USA 3 Division of Biophysical Chemistry, Biozentrum, Uni- versity of Basel, Klingelbergstr. 70, CH-4056 Basel, Switzerland Aquaporin-0 (AQP0) is the major membrane protein in vertebrate eye lenses. It has been proposed that AQP0 tetramers mediate contact between membranes of adjacent lens fiber cells, which would be consistent with the extraordinarily narrow inter-cellular spacing. We have obtained 3D crystals of recombinant bovine AQP0 that diffract to 7.0 A ˚ resolution. The crystal packing was determined by molecular replacement and shows that, within the cubic lattice, AQP0 tetramers are associated head-to-head along their 4-fold axes. Oligomeric states larger than the tetramer were also observed in solution by native gel electrophoresis and analytical ultracentrifugation methods. In the crystals, there are no direct contacts between octamers, and it can thus be inferred that crystalline order is mediated solely by the detergent belts surrounding the membrane protein. Across the tetramer–tetramer interface, extracellular loops A and C interdigitate at the center and the perimeter of the octamer, respectively. The octamer structure is compared with that of the recently determined structure of truncated ovine AQP0 derived from electron diffraction of 2D crystals. Intriguingly, also in these crystals, octamers are observed, but with significantly different relative tetramer–tetramer orientations. The interactions observed in the loosely packed 3D crystals reported here may in fact represent an in vivo association mode between AQP0 tetramers from juxtaposed membranes in the eye lens. q 2005 Elsevier Ltd. All rights reserved. Keywords: eye lens; MIP26; X-ray crystallography; membrane protein; mixed micelle *Corresponding author The major intrinsic protein (MIP26, AQP0), an integral membrane protein present in vertebrate eye lenses, 1 is the founding member of the aquaporin superfamily. 2,3 AQP0 is the most abundant protein in the plasma membrane of lens fiber cells constituting almost 50% of the total protein. 4 There is extensive intercellular coupling in this tissue, which renders AQP0 a possible candidate to mediate cell–cell interactions. 5 Knock-out of this protein in Cat Fr mice 6 causes cataract formation. Similarly, mutations in AQP0 that prevent proper folding or targeting cause lens opacification sup- porting a crucial role of AQP0 in the development of the transparent lens. 7 Water channel function of AQP0 (though about 40 times less efficient compared to AQP1 8 ) and facilitation of glycerol translocation 9 have been demonstrated for AQP0, but its putative role in mediating cell–cell inter- actions appears unique amongst aquaporins. The arrangement of AQP0 in native membranes has been examined in freeze and label-fractured preparations by electron microscopy using immuno- labeling. 10,11 AQP0 was found only in tightly abutting membranes (thin and wavy junctions) but not in thick junctions. 10 Bok et al. were able to show close interaction between membrane patches that contained AQP0 on both sides. 11 This was confirmed later by analogous studies on AQP0 containing proteoliposomes. 12 In another study, however, no interaction between proteoliposomes, but only between proteoliposomes and phosphatidylserine liposomes was demonstrated employing energy 0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. Abbreviations used: AQP, Aquaporin; CMC, critical micellar concentration; OG, n-octyl-b-D-glucoside; DM, n-decyl-b-D-maltoside; DDM, n-dodecyl-b-D-maltoside; NG, n-nonyl-b-D-glucoside; EM, electron microscope; TLS, translation-libration-screw. E-mail address of the corresponding author: tilman.schirmer@unibas.ch doi:10.1016/j.jmb.2005.10.032 J. Mol. Biol. (2006) 355, 605–611