Structural models of the supramolecular organization of AQP0 and connexons in junctional microdomains Simon Scheuring a, * , Nikolay Buzhynskyy a , Szymon Jaroslawski a , Rui Pedro Gonc ¸alves a , Richard K. Hite b , Thomas Walz b a Institut Curie, UMR168-CNRS, 26 Rue d’Ulm, 75248 Paris, France b Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA Received 1 April 2007; received in revised form 15 June 2007; accepted 6 July 2007 Available online 28 July 2007 Abstract Membrane proteins perform many essential cellular functions. Over the last years, substantial advances have been made in our under- standing of the structure and function of isolated membrane proteins. However, like soluble proteins, many membrane proteins assemble into supramolecular complexes that perform specific functions in specialized membrane domains. Since supramolecular complexes of membrane proteins are difficult to study by conventional approaches, little is known about their composition, organization and assem- bly. The high signal-to-noise ratio of the images that can be obtained with an atomic force microscope (AFM) makes this instrument a powerful tool to image membrane protein complexes within native membranes. Recently, we have reported high-resolution topographs of junctional microdomains in native eye lens membranes containing two-dimensional (2D) arrays of aquaporin-0 (AQP0) surrounded by connexons. While both proteins are involved in cell adhesion, AQP0 is a specific water channel whereas connexons form cell–cell com- munication channels with broad substrate specificity. Here, we have performed a detailed analysis of the supramolecular organization of AQP0 tetramers and connexon hexamers in junctional microdomains in the native lens membrane. We present first structural models of these junctional microdomains, which we generated by docking atomic models of AQP0 and connexons into the AFM topographs. The AQP0 2D arrays in the native membrane show the same molecular packing of tetramers seen in highly ordered double-layered 2D crys- tals obtained through reconstitution of purified AQP0. In contrast, the connexons that surround the AQP0 arrays are only loosely packed. Based on our AFM observations, we propose a mechanism that may explain the supramolecular organization of AQP0 and connexons in junctional domains in native lens membranes. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Aquaporin; Atomic force microscopy; Connexin; Membrane structure; Supramolecular organization 1. Introduction Membrane proteins play key roles in many vital cellular pro- cesses, such as energy conversion, signal transduction, and cell adhesion. Structural analysis of purified membrane proteins by X-ray crystallography of three-dimensional (3D 1 ) crystals and electron crystallography of 2D crystals has greatly advanced our understanding of membrane processes. However, the 120 membrane protein structures known to date belong to only about 30 families of membrane proteins (Michel, 2007; White, 2007). Moreover, most membrane proteins are thought to be rather densely packed in membranes (Eng- elman, 2005) and to work in ensembles or even in functional supercomplexes (Joliot et al., 1989; Scha ¨gger and Pfeiffer, 2001; Scheuring, 2006). The atomic force microscope (AFM; Binnig et al., 1986) can record images with an excep- tionally high signal-to-noise ratio, making it a unique tool that can provide structural information of individual 1047-8477/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2007.07.009 * Corresponding author. Fax: +33 1 40510636. E-mail address: simon.scheuring@curie.fr (S. Scheuring). 1 Abbreviations used: 2D, two-dimensional; 3D, three-dimensional; AFM, atomic force microscopy; AQP0, aquaporin-0; Cx, connexin; EM, electron microscopy. www.elsevier.com/locate/yjsbi Available online at www.sciencedirect.com Journal of Structural Biology 160 (2007) 385–394 Journal of Structural Biology