Homology modeling of representative subfamilies of Arabidopsis major intrinsic proteins. Classification based on the aromatic/arginine selectivity filter

Abstract

Major intrinsic proteins (MIPs) are a family of membrane channels that facilitate the bidirectional transport of water and small uncharged solutes such as glycerol. The 35 full-length members of the MIP family in Arabidopsis are segregated into four structurally homologous subfamilies: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin 26-like intrinsic membrane proteins (NIPs), and small basic intrinsic proteins (SIPs). Computational methods were used to construct structural models of the putative pore regions of various plant MIPs based on homology modeling with the atomic resolution crystal structures of mammalian aquaporin 1 and the bacterial glycerol permease GlpF. Based on comparisons of the narrow selectivity filter regions (the aromatic/Arg [ar/R] filter), the members of the four phylogenetic subfamilies of Arabidopsis MIPs can be classified into eight groups. PIPs possess a uniform ar/R signature characteristic of high water transport aquaporins, whereas TIPs are highly diverse with three separate conserved ar/R regions. NIPs possess two separate conserved ar/R regions, one that is similar to the archetype, soybean (Glycine max) nodulin 26, and another that is characteristic of Arabidopsis NIP6;1. The SIP subfamily possesses two ar/R subgroups, characteristic of either SIP1 or SIP2. Both SIP ar/R residues are divergent from all other MIPs in plants and other kingdoms. Overall, these findings suggest that higher plant MIPs have a common fold but show distinct differences in proposed pore apertures, potential to form hydrogen bonds with transported molecules, and amphiphilicity that likely results in divergent transport selectivities.

Figure 1.

Topology of MIPs and architecture of the ar/R selectivity region. A, The general topology conserved in the MIP family is shown. Each NPA half-repeat is color coordinated with transmembrane α-helices 1 to 3 shown and the first NPA half-helix (loop B) shown in blue and yellow, respectively, and transmembrane α-helices 4 to 6 and the second NPA half helix (loop E) shown in red and white, respectively. The relative positions of the four residues on H2, H5, and loop E (LE₁ and LE₂) that comprise the ar/R tetrad are indicated. B, The assembly of these structural elements into the MIP hourglass fold is shown using the crystal structure of bovine AQP1 (Sui et al., 2001). Three of the four waters traversing the AQP1 pore are shown as space-filling aqua spheres. The extracellular (Ex) and cytosolic (Cyto) sides are indicated, and the position of the lipid bilayer is shown as a solid bar. C, Representation of the ar/R and NPA constrictions in the AQP1 pore showing the disposition of the ar/R tetrad (magenta) and the NPA Asn side chains (white and yellow) relative to waters traversing the pore. D, Comparison of the ar/R selectivity filters of AQP1 and the glyceroporin GlpF (Fu et al., 2000) with conductant (water or glycerol) bound. Residue side chains are colored blue for basic hydrophilic, yellow for hydrophobic, and white for neutral hydrophilic. For glycerol, the hydrocarbon backbone is magenta, and the hydroxyl groups are aqua.

Figure 2.

Structural analysis of the nodulin 26 homology model. A, Superposition of the experimental AQP1 structure (red) and the nodulin 26 homology model (green) is shown viewed perpendicular to the pore of the protein. B, The superposed positions of the ar/R tetrad for AQP1 (red) and nodulin 26 (green) are shown viewed perpendicular to the plane of the membrane. The positions of the ar/R residues from H2, H5, and loop E (LE₁ and LE₂) are indicated.

Figure 3.

Comparison of the ar/R selectivity regions of the NIP and PIP subfamilies. Space-filling side-chain residues of the ar/R selectivity region of AtPIP1;1, soybean nodulin 26, and AtNIP6;1 shown compared to AQP1 and GlpF with conductant bound. The single-letter amino acid code appears along side each residue, and residue side chains are colored blue for basic hydrophilic, yellow for hydrophobic, and white for neutral hydrophilic. The ar/R filter in each projection is viewed perpendicular to the plane of the bilayer from the extracellular vestibule.

Figure 4.

Analysis of the putative pore dimensions of representative plant MIPs compared with AQP1. The dimensions of the pore regions of AQP1 and the molecular models of nodulin 26, NIP6;1, TIP3:1, and TIP5;1 were analyzed by using the HOLE program (Smart et al., 1993) and were visualized on InsightII. The pores are viewed perpendicular to the pore axis and are labeled with respect to the location of the ar/R (A) and NPA (N) regions.

Figure 5.

Comparison of the ar/R selectivity regions of the TIP and SIP subfamilies. Space-filling side-chain residues of the ar/R selectivity region are shown from homology models of representative subgroups of the TIP and the SIP subfamilies. For comparison, the ar/R region of nodulin 26 is included. Residues are color coordinated according to charge and hydrophilicity as described in the legend for Figure 1.