Projection map of aquaporin-9 at 7 A resolution

Abstract

Aquaporin-9, an aquaglyceroporin present in diverse tissues, is unique among aquaporins because it is not only permeable to water, urea and glycerol, but also allows passage of larger uncharged solutes. Single particle analysis of negatively stained recombinant rat aquaporin-9 revealed a particle size characteristic of the tetrameric organization of all members of the aquaporin family. Reconstitution of aquaporin-9 into two-dimensional crystals enabled us to calculate a projection map at 7 A resolution. The projection structure indicates a tetrameric structure, similar to GlpF, with each square-like monomer forming a pore. A comparison of the pore-lining residues between the crystal structure of GlpF and a homology model of aquaporin-9 locates substitutions in these residues predominantly to the hydrophobic edge of the tripathic pore of GlpF, providing first insights into the structural basis for the broader substrate specificity of aquaporin-9.

Figure 1

Protein purification and single particle EM analysis. (a) Silver stained 10% SDS PAGE gel of purified recombinant rat AQP9 before (lane 1) and after deglycosylation with PNGase F (lane 2). (b) Image of negatively stained AQP9 showing that the OG-solubilized particles are mono-dispersed and homogeneous in size. The scale bar is 100 nm. (c) Representative single particle averages. The side length of the individual panels is 50 nm.

Figure 2

Analysis of AQP9 2D crystals. (a) The image of a negatively stained AQP9 2D crystal clearly reveals the square crystal lattice. Scale bar is 200 nm. (b) Fourier transform of the image shown in (a). Scale bar is 5 nm⁻¹. (c) CTF plot of an image of a glucose-embedded 2D crystal. The circles represent CTF zero transitions and the boxed numbers depict the IQ values of the individual reflections as defined by . Scale bar is 1.7 nm⁻¹. (d) p42₁2-symmetrized projection map of AQP9 at 7 Å resolution. A unit cell, lattice dimensions a = b = 103.0 Å, γ = 90º, contains two AQP9 tetramers and is outlined in black. Scale bar is 50 nm.

Figure 3

Electron diffraction of an AQP9 2D crystal. The diffraction pattern was recorded from a glucose-embedded crystal and shows diffraction spots to a resolution of about 3.8 Å. Scale bar is 1 nm⁻¹.

Figure 4

Projection maps of different aquaporins. (a) Projection structure of the AQP9 tetramer at 7 Å resolution. Asterisk indicates the weaker density of the AQP9 pore as compared to the pore in GlpF. (b) Projection structure of GlpF at 3.7 Å resolution. Reprinted with permission from . (c) Projection structure of AQP0 at 4 Å resolution (adapted from 38). Scale bars are 25 nm.

Figure 5

Multiple sequence alignment. Multiple sequence alignment of rat, human and mouse AQP9 with GlpF, AQP1, AQP0 and AQPZ (aquaporins with known 3D structures). Secondary structure assignments are shown according to the crystal structure of GlpF . Residue numbering follows the rat AQP9 sequence. Residues in red are conserved in all aquaporins, residues in green are only conserved in glycerol-conducting aquaporins, and residues in blue are only conserved in AQP9 homologs. Residues marked with a “P” form part of the pore in the GlpF structure, according to the program HOLE . A green “P” indicates a pore residue in GlpF that is conserved in all aquaporins, a blue “P” a pore residue in GlpF that is conserved in all aquaglyceroporins, a red “P” a pore residue in GlpF that is different from all the AQP9 sequences shown, and an uncolored “P” a pore residue in GlpF that is identical to one or two AQP9 sequences.

Figure 6

Conservation of pore residues in aquaporins. (a)–(c) Conserved pore residues shown in a homology model for AQP9. Residues conserved in all aquaporins are shown in green (a), those only conserved in glycerol-conducting aquaporins in blue (b), and those that differ between GlpF and AQP9 homologs in red (c).