Three-dimensional fold of the human AQP1 water channel determined at 4 Å resolution by electron crystallography of two-dimensional crystals embedded in ice

Abstract

Here, we present a three-dimensional (3D) density map of deglycosylated, human erythrocyte aquaporin 1 (AQP1) determined at 4 Å resolution in plane and ∼7 Å resolution perpendicular to the bilayer. The map was calculated by analyzing images and electron diffraction patterns recorded from tilted (up to 60 °), ice-embedded, frozen-hydrated 2D crystals of AQP1 in lipid bilayer membranes. This map significantly extends the findings related to the folding of the AQP1 polypeptide chain determined by us at a lower, 7 Å by ∼20 Å, resolution. The solvent-accessible volume within a monomer has a vestibular architecture, with a narrow, ∼6.5 Å diameter constriction near the center of the bilayer, where the location of the water-selective channel is postulated to exist. The clearly resolved densities for the transmembrane helices display the protrusions expected for bulky side-chains. The density in the interior of the helix barrel (putative NPA box region) is better resolved compared to our previous map, suggesting clearer linkage to some of the helices, and it may harbor short stretches of α-helix. At the bilayer extremities, densities for some of the inter-helix hydrophilic loops are visible. Consistent with these observed inter-helix connections, possible models for the threading of the AQP1 polypeptide chain are presented. A preferred model is deduced that agrees with the putative locations of a group of aromatic residues in the amino acid sequence and in the 3D density map.