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Crystal Structure of an Ammonia-Permeable Aquaporin.

Kirscht A, Kaptan SS, Bienert GP, Chaumont F, Nissen P, de Groot BL, Kjellbom P, Gourdon P, Johanson U - PLoS Biol. (2016)

Bottom Line: By mutational studies, we show that the identified determinants in the extended selectivity filter region are sufficient to convert a strictly water-specific human aquaporin into an AtTIP2;1-like ammonia channel.A flexible histidine and a novel water-filled side pore are speculated to deprotonate ammonium ions, thereby possibly increasing permeation of ammonia.The molecular understanding of how aquaporins facilitate ammonia flux across membranes could potentially be used to modulate ammonia losses over the plasma membrane to the atmosphere, e.g., during photorespiration, and thereby to modify the nitrogen use efficiency of plants.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Structural Biology, Center for Molecular Protein Science, Lund University, Lund, Sweden.

ABSTRACT
Aquaporins of the TIP subfamily (Tonoplast Intrinsic Proteins) have been suggested to facilitate permeation of water and ammonia across the vacuolar membrane of plants, allowing the vacuole to efficiently sequester ammonium ions and counteract cytosolic fluctuations of ammonia. Here, we report the structure determined at 1.18 Å resolution from twinned crystals of Arabidopsis thaliana aquaporin AtTIP2;1 and confirm water and ammonia permeability of the purified protein reconstituted in proteoliposomes as further substantiated by molecular dynamics simulations. The structure of AtTIP2;1 reveals an extended selectivity filter with the conserved arginine of the filter adopting a unique unpredicted position. The relatively wide pore and the polar nature of the selectivity filter clarify the ammonia permeability. By mutational studies, we show that the identified determinants in the extended selectivity filter region are sufficient to convert a strictly water-specific human aquaporin into an AtTIP2;1-like ammonia channel. A flexible histidine and a novel water-filled side pore are speculated to deprotonate ammonium ions, thereby possibly increasing permeation of ammonia. The molecular understanding of how aquaporins facilitate ammonia flux across membranes could potentially be used to modulate ammonia losses over the plasma membrane to the atmosphere, e.g., during photorespiration, and thereby to modify the nitrogen use efficiency of plants.

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Related in: MedlinePlus

Pore diameter and the extended selectivity filter.(A) Individual profiles of AtTIP2;1 (green), glycerol-permeable EcGlpF (blue), water-specific SoPIP2;1 (closed conformation; purple), and HsAQP4 (orange), as well as average diameter of five other open water-specific AQP structures (dashed line). Protein Data Bank IDs are provided in S1 Table. NPA region and selectivity filter (SF) indicated by shading. In contrast to previously reported structures of AQPs, where the SF region constitutes the most narrow part of the channel, the pore diameter of AtTIP2;1 is more uniform throughout the channel. (B) Graphic representation of a side view of the AtTIP2;1 pore aligned with (A). The selectivity filter is highlighted by stick representation of residues in positions H2P, HEP, and LCP (left to right). Nondisplayed residues at positions H5P and LEP are located in front of the visual plane. Close-up depicts electron density at 4σ. The high resolution of the structure makes it possible to pinpoint the nitrogen atoms in imidazole rings of histidines. The Nε of LCP-His 131 forms a hydrogen bond (dashed yellow line) to a water molecule (Wat2) in the pore. (C) Vacuolar (top view of AtTIP2;1) and corresponding extracellular view (SoPIP2;1 and EcGlpF) on the amino acid residues at the five positions (H2P, LCP, H5P, LEP, and HEP) comprising the extended selectivity filter of the pore. AtTIP2;1 (green) is compared to the water-specific SoPIP2;1 (purple) and the glycerol-permeable EcGlpF (blue). In AtTIP2;1, histidines at H2P and LCP stabilize the arginine (Arg 200) at HEP in a novel orientation, which is clearly different from its positioning in structures of water-specific and glycerol-permeable AQPs. The spatial orientation of the backbone carbonyls at position LEP is similar in AtTIP2;1 and EcGlpF, whereas it deviates in the water-specific SoPIP2;1. The Ile 185 at H5P of AtTIP2;1 results in a wider SF region compared to water-specific AQPs that have a histidine at this position. (D) The conservation of residues in the extended selectivity filter displayed in (C). The LCP position that extends the selectivity filter is boxed in red. Plant TIPs and mammalian AQP8s are similar and distinctly different from water-specific AQPs in animals and plants (PIPs, plasma membrane intrinsic proteins), as well as the glycerol channel GlpF. Conservation patterns suggest a similar orientation of the conserved arginine at position HEP of the selectivity filter of all TIPs and AQP8s, and furthermore that individual subgroups of TIPs and water-specific AQPs might have evolved specialized substrate profiles (details in S2 Table). Asterisk denotes identity to TIP2s, and colors highlight selectivity filters shown in (C).
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pbio.1002411.g003: Pore diameter and the extended selectivity filter.(A) Individual profiles of AtTIP2;1 (green), glycerol-permeable EcGlpF (blue), water-specific SoPIP2;1 (closed conformation; purple), and HsAQP4 (orange), as well as average diameter of five other open water-specific AQP structures (dashed line). Protein Data Bank IDs are provided in S1 Table. NPA region and selectivity filter (SF) indicated by shading. In contrast to previously reported structures of AQPs, where the SF region constitutes the most narrow part of the channel, the pore diameter of AtTIP2;1 is more uniform throughout the channel. (B) Graphic representation of a side view of the AtTIP2;1 pore aligned with (A). The selectivity filter is highlighted by stick representation of residues in positions H2P, HEP, and LCP (left to right). Nondisplayed residues at positions H5P and LEP are located in front of the visual plane. Close-up depicts electron density at 4σ. The high resolution of the structure makes it possible to pinpoint the nitrogen atoms in imidazole rings of histidines. The Nε of LCP-His 131 forms a hydrogen bond (dashed yellow line) to a water molecule (Wat2) in the pore. (C) Vacuolar (top view of AtTIP2;1) and corresponding extracellular view (SoPIP2;1 and EcGlpF) on the amino acid residues at the five positions (H2P, LCP, H5P, LEP, and HEP) comprising the extended selectivity filter of the pore. AtTIP2;1 (green) is compared to the water-specific SoPIP2;1 (purple) and the glycerol-permeable EcGlpF (blue). In AtTIP2;1, histidines at H2P and LCP stabilize the arginine (Arg 200) at HEP in a novel orientation, which is clearly different from its positioning in structures of water-specific and glycerol-permeable AQPs. The spatial orientation of the backbone carbonyls at position LEP is similar in AtTIP2;1 and EcGlpF, whereas it deviates in the water-specific SoPIP2;1. The Ile 185 at H5P of AtTIP2;1 results in a wider SF region compared to water-specific AQPs that have a histidine at this position. (D) The conservation of residues in the extended selectivity filter displayed in (C). The LCP position that extends the selectivity filter is boxed in red. Plant TIPs and mammalian AQP8s are similar and distinctly different from water-specific AQPs in animals and plants (PIPs, plasma membrane intrinsic proteins), as well as the glycerol channel GlpF. Conservation patterns suggest a similar orientation of the conserved arginine at position HEP of the selectivity filter of all TIPs and AQP8s, and furthermore that individual subgroups of TIPs and water-specific AQPs might have evolved specialized substrate profiles (details in S2 Table). Asterisk denotes identity to TIP2s, and colors highlight selectivity filters shown in (C).

Mentions: Interestingly, the pore diameter of AtTIP2;1 at the NPA region is smaller than in other AQPs, and it remains constant at around 3 Å throughout the pore (Fig 3A and 3B). This is unusual, since in other structures of open AQPs, the aromatic/arginine selectivity filter constitutes the narrowest part of the pore. As mentioned earlier, amino acid residues at the four positions of the pore selectivity filter in helix 2, helix 5, loop E, and helix E (specifically denoted H2P, H5P, LEP, and HEP) are thought to determine the substrate specificity (Figs 1A and 3C). In line with this, TIP2s deviate from other AQPs (Fig 3D), and as expected from mutational studies and modeling [2,22], the wider selectivity filter is mainly due to an isoleucine (Ile 185) at position H5P in helix 5, replacing a histidine that is conserved in the water-specific AQPs. However, the most striking feature of the AtTIP2;1 selectivity filter arises from an unpredicted positioning of the arginine at HEP in helix E (Arg 200), a conserved residue in nearly all AQPs. In AtTIP2;1, the arginine side chain is pushed to the side of the pore by a histidine located in loop C (His 131), which now appears as a fifth residue (LCP) of an extended selectivity filter. The novel position of the arginine is further stabilized by a hydrogen bond to the histidine (His 63) at position H2P in helix 2, which occupies essentially the same space as corresponding aromatic residues of water and glycerol channels (e.g., Phe 81 in SoPIP2;1 [14], Trp 48 in EcGlpF [12]) without direct effects on the pore aperture. The close interaction with Arg 200 at position HEP in helix E suggests a shift in the pKa of His 63 at position H2P, which is likely to stay unprotonated also in the acidic environment of the vacuole. In contrast to His 63, the additional His 131 at position LCP in loop C points to the center of the pore and forms a hydrogen bond to a pore-water (Wat 2; Fig 3B). Hence, AtTIP2;1 represents the first AQP structure where a residue in loop C (His 131) directly participates in interactions with the substrate in the selectivity region, defining an extended selectivity filter with five positions. The histidine residue at position H2P in helix 2 is conserved in all TIPs, whereas the histidine at position LCP in loop C is only maintained in some types of TIPs, including the TIP2 isoforms, and appears to have been replaced by phenylalanine in a common ancestor of TIP1s and TIP3s (Fig 3D) [5]. A phenylalanine at position LCP in loop C is also capable of sterically directing the arginine at position HEP in helix E to the side of the pore, but provides a more hydrophobic environment at the selectivity filter. Worth noting, similar to TIP3s, the mammalian AQP8s [9,10] also possess a histidine at position H2P in helix 2, lack a conserved histidine in loop C, and can be aligned with a phenylalanine at position LCP in loop C (Fig 3D). Thus, a histidine at position H2P in helix 2 and an aromatic residue at position LCP in loop C seem to be a common feature among ammonia-permeable AQPs both in plants and animals. This suggests that the derived phenylalanine at LCP in loop C of some plant TIPs, which supports ammonia permeability without the ability to form hydrogen bonds to the substrate, reflects an adaptation to a different milieu, e.g., regarding pH or alternatively altered requirements on permeation rate and selectivity.


Crystal Structure of an Ammonia-Permeable Aquaporin.

Kirscht A, Kaptan SS, Bienert GP, Chaumont F, Nissen P, de Groot BL, Kjellbom P, Gourdon P, Johanson U - PLoS Biol. (2016)

Pore diameter and the extended selectivity filter.(A) Individual profiles of AtTIP2;1 (green), glycerol-permeable EcGlpF (blue), water-specific SoPIP2;1 (closed conformation; purple), and HsAQP4 (orange), as well as average diameter of five other open water-specific AQP structures (dashed line). Protein Data Bank IDs are provided in S1 Table. NPA region and selectivity filter (SF) indicated by shading. In contrast to previously reported structures of AQPs, where the SF region constitutes the most narrow part of the channel, the pore diameter of AtTIP2;1 is more uniform throughout the channel. (B) Graphic representation of a side view of the AtTIP2;1 pore aligned with (A). The selectivity filter is highlighted by stick representation of residues in positions H2P, HEP, and LCP (left to right). Nondisplayed residues at positions H5P and LEP are located in front of the visual plane. Close-up depicts electron density at 4σ. The high resolution of the structure makes it possible to pinpoint the nitrogen atoms in imidazole rings of histidines. The Nε of LCP-His 131 forms a hydrogen bond (dashed yellow line) to a water molecule (Wat2) in the pore. (C) Vacuolar (top view of AtTIP2;1) and corresponding extracellular view (SoPIP2;1 and EcGlpF) on the amino acid residues at the five positions (H2P, LCP, H5P, LEP, and HEP) comprising the extended selectivity filter of the pore. AtTIP2;1 (green) is compared to the water-specific SoPIP2;1 (purple) and the glycerol-permeable EcGlpF (blue). In AtTIP2;1, histidines at H2P and LCP stabilize the arginine (Arg 200) at HEP in a novel orientation, which is clearly different from its positioning in structures of water-specific and glycerol-permeable AQPs. The spatial orientation of the backbone carbonyls at position LEP is similar in AtTIP2;1 and EcGlpF, whereas it deviates in the water-specific SoPIP2;1. The Ile 185 at H5P of AtTIP2;1 results in a wider SF region compared to water-specific AQPs that have a histidine at this position. (D) The conservation of residues in the extended selectivity filter displayed in (C). The LCP position that extends the selectivity filter is boxed in red. Plant TIPs and mammalian AQP8s are similar and distinctly different from water-specific AQPs in animals and plants (PIPs, plasma membrane intrinsic proteins), as well as the glycerol channel GlpF. Conservation patterns suggest a similar orientation of the conserved arginine at position HEP of the selectivity filter of all TIPs and AQP8s, and furthermore that individual subgroups of TIPs and water-specific AQPs might have evolved specialized substrate profiles (details in S2 Table). Asterisk denotes identity to TIP2s, and colors highlight selectivity filters shown in (C).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4814140&req=5

pbio.1002411.g003: Pore diameter and the extended selectivity filter.(A) Individual profiles of AtTIP2;1 (green), glycerol-permeable EcGlpF (blue), water-specific SoPIP2;1 (closed conformation; purple), and HsAQP4 (orange), as well as average diameter of five other open water-specific AQP structures (dashed line). Protein Data Bank IDs are provided in S1 Table. NPA region and selectivity filter (SF) indicated by shading. In contrast to previously reported structures of AQPs, where the SF region constitutes the most narrow part of the channel, the pore diameter of AtTIP2;1 is more uniform throughout the channel. (B) Graphic representation of a side view of the AtTIP2;1 pore aligned with (A). The selectivity filter is highlighted by stick representation of residues in positions H2P, HEP, and LCP (left to right). Nondisplayed residues at positions H5P and LEP are located in front of the visual plane. Close-up depicts electron density at 4σ. The high resolution of the structure makes it possible to pinpoint the nitrogen atoms in imidazole rings of histidines. The Nε of LCP-His 131 forms a hydrogen bond (dashed yellow line) to a water molecule (Wat2) in the pore. (C) Vacuolar (top view of AtTIP2;1) and corresponding extracellular view (SoPIP2;1 and EcGlpF) on the amino acid residues at the five positions (H2P, LCP, H5P, LEP, and HEP) comprising the extended selectivity filter of the pore. AtTIP2;1 (green) is compared to the water-specific SoPIP2;1 (purple) and the glycerol-permeable EcGlpF (blue). In AtTIP2;1, histidines at H2P and LCP stabilize the arginine (Arg 200) at HEP in a novel orientation, which is clearly different from its positioning in structures of water-specific and glycerol-permeable AQPs. The spatial orientation of the backbone carbonyls at position LEP is similar in AtTIP2;1 and EcGlpF, whereas it deviates in the water-specific SoPIP2;1. The Ile 185 at H5P of AtTIP2;1 results in a wider SF region compared to water-specific AQPs that have a histidine at this position. (D) The conservation of residues in the extended selectivity filter displayed in (C). The LCP position that extends the selectivity filter is boxed in red. Plant TIPs and mammalian AQP8s are similar and distinctly different from water-specific AQPs in animals and plants (PIPs, plasma membrane intrinsic proteins), as well as the glycerol channel GlpF. Conservation patterns suggest a similar orientation of the conserved arginine at position HEP of the selectivity filter of all TIPs and AQP8s, and furthermore that individual subgroups of TIPs and water-specific AQPs might have evolved specialized substrate profiles (details in S2 Table). Asterisk denotes identity to TIP2s, and colors highlight selectivity filters shown in (C).
Mentions: Interestingly, the pore diameter of AtTIP2;1 at the NPA region is smaller than in other AQPs, and it remains constant at around 3 Å throughout the pore (Fig 3A and 3B). This is unusual, since in other structures of open AQPs, the aromatic/arginine selectivity filter constitutes the narrowest part of the pore. As mentioned earlier, amino acid residues at the four positions of the pore selectivity filter in helix 2, helix 5, loop E, and helix E (specifically denoted H2P, H5P, LEP, and HEP) are thought to determine the substrate specificity (Figs 1A and 3C). In line with this, TIP2s deviate from other AQPs (Fig 3D), and as expected from mutational studies and modeling [2,22], the wider selectivity filter is mainly due to an isoleucine (Ile 185) at position H5P in helix 5, replacing a histidine that is conserved in the water-specific AQPs. However, the most striking feature of the AtTIP2;1 selectivity filter arises from an unpredicted positioning of the arginine at HEP in helix E (Arg 200), a conserved residue in nearly all AQPs. In AtTIP2;1, the arginine side chain is pushed to the side of the pore by a histidine located in loop C (His 131), which now appears as a fifth residue (LCP) of an extended selectivity filter. The novel position of the arginine is further stabilized by a hydrogen bond to the histidine (His 63) at position H2P in helix 2, which occupies essentially the same space as corresponding aromatic residues of water and glycerol channels (e.g., Phe 81 in SoPIP2;1 [14], Trp 48 in EcGlpF [12]) without direct effects on the pore aperture. The close interaction with Arg 200 at position HEP in helix E suggests a shift in the pKa of His 63 at position H2P, which is likely to stay unprotonated also in the acidic environment of the vacuole. In contrast to His 63, the additional His 131 at position LCP in loop C points to the center of the pore and forms a hydrogen bond to a pore-water (Wat 2; Fig 3B). Hence, AtTIP2;1 represents the first AQP structure where a residue in loop C (His 131) directly participates in interactions with the substrate in the selectivity region, defining an extended selectivity filter with five positions. The histidine residue at position H2P in helix 2 is conserved in all TIPs, whereas the histidine at position LCP in loop C is only maintained in some types of TIPs, including the TIP2 isoforms, and appears to have been replaced by phenylalanine in a common ancestor of TIP1s and TIP3s (Fig 3D) [5]. A phenylalanine at position LCP in loop C is also capable of sterically directing the arginine at position HEP in helix E to the side of the pore, but provides a more hydrophobic environment at the selectivity filter. Worth noting, similar to TIP3s, the mammalian AQP8s [9,10] also possess a histidine at position H2P in helix 2, lack a conserved histidine in loop C, and can be aligned with a phenylalanine at position LCP in loop C (Fig 3D). Thus, a histidine at position H2P in helix 2 and an aromatic residue at position LCP in loop C seem to be a common feature among ammonia-permeable AQPs both in plants and animals. This suggests that the derived phenylalanine at LCP in loop C of some plant TIPs, which supports ammonia permeability without the ability to form hydrogen bonds to the substrate, reflects an adaptation to a different milieu, e.g., regarding pH or alternatively altered requirements on permeation rate and selectivity.

Bottom Line: By mutational studies, we show that the identified determinants in the extended selectivity filter region are sufficient to convert a strictly water-specific human aquaporin into an AtTIP2;1-like ammonia channel.A flexible histidine and a novel water-filled side pore are speculated to deprotonate ammonium ions, thereby possibly increasing permeation of ammonia.The molecular understanding of how aquaporins facilitate ammonia flux across membranes could potentially be used to modulate ammonia losses over the plasma membrane to the atmosphere, e.g., during photorespiration, and thereby to modify the nitrogen use efficiency of plants.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Structural Biology, Center for Molecular Protein Science, Lund University, Lund, Sweden.

ABSTRACT
Aquaporins of the TIP subfamily (Tonoplast Intrinsic Proteins) have been suggested to facilitate permeation of water and ammonia across the vacuolar membrane of plants, allowing the vacuole to efficiently sequester ammonium ions and counteract cytosolic fluctuations of ammonia. Here, we report the structure determined at 1.18 Å resolution from twinned crystals of Arabidopsis thaliana aquaporin AtTIP2;1 and confirm water and ammonia permeability of the purified protein reconstituted in proteoliposomes as further substantiated by molecular dynamics simulations. The structure of AtTIP2;1 reveals an extended selectivity filter with the conserved arginine of the filter adopting a unique unpredicted position. The relatively wide pore and the polar nature of the selectivity filter clarify the ammonia permeability. By mutational studies, we show that the identified determinants in the extended selectivity filter region are sufficient to convert a strictly water-specific human aquaporin into an AtTIP2;1-like ammonia channel. A flexible histidine and a novel water-filled side pore are speculated to deprotonate ammonium ions, thereby possibly increasing permeation of ammonia. The molecular understanding of how aquaporins facilitate ammonia flux across membranes could potentially be used to modulate ammonia losses over the plasma membrane to the atmosphere, e.g., during photorespiration, and thereby to modify the nitrogen use efficiency of plants.

Show MeSH
Related in: MedlinePlus