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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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MD simulations of AtTIP2;1.(A) The potential mean force (PMF) profiles for ammonia through AtTIP2;1 (red) and through a model membrane containing 20% cholesterol (green). In the lower part of the panel, the number of hydrogen bonds between ammonia and AtTIP2;1 are shown as function of position along the pore axis. Interactions with residues in the extended selectivity filter depicted in (C) are color-coded to resolve their contribution (H2P-His 63 (blue), LCP-His 131 (red), LEP-Gly 194 (green) and HEP-Arg 200 (brown)), and demonstrate hydrogen bonding to each of the four polar residues of the extended selectivity filter. (B) Snapshots of ammonia permeation. Cross section of AtTIP2;1 shown as grey surface and green cartoon of the backbone. Side chains of selected amino acid residues in the selectivity filter are displayed as sticks and color coded as in (C). (C) Close-up of an ammonia molecule at the center, forming hydrogen bonds to four residues (H2P-His 63, LCP-His 131, LEP-Gly 194, and HEP-Arg 200) of the selectivity filter. The hydrogen bonds are indicated by orange dashes and distances are given in Å. Ile 185 at position H5P of the selectivity filter, located in front of the visual plane, is not shown. The underlying data of panel A can be found in S1 Data.
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pbio.1002411.g006: MD simulations of AtTIP2;1.(A) The potential mean force (PMF) profiles for ammonia through AtTIP2;1 (red) and through a model membrane containing 20% cholesterol (green). In the lower part of the panel, the number of hydrogen bonds between ammonia and AtTIP2;1 are shown as function of position along the pore axis. Interactions with residues in the extended selectivity filter depicted in (C) are color-coded to resolve their contribution (H2P-His 63 (blue), LCP-His 131 (red), LEP-Gly 194 (green) and HEP-Arg 200 (brown)), and demonstrate hydrogen bonding to each of the four polar residues of the extended selectivity filter. (B) Snapshots of ammonia permeation. Cross section of AtTIP2;1 shown as grey surface and green cartoon of the backbone. Side chains of selected amino acid residues in the selectivity filter are displayed as sticks and color coded as in (C). (C) Close-up of an ammonia molecule at the center, forming hydrogen bonds to four residues (H2P-His 63, LCP-His 131, LEP-Gly 194, and HEP-Arg 200) of the selectivity filter. The hydrogen bonds are indicated by orange dashes and distances are given in Å. Ile 185 at position H5P of the selectivity filter, located in front of the visual plane, is not shown. The underlying data of panel A can be found in S1 Data.

Mentions: Although the high resolution structure of AtTIP2;1 allowed us to discriminate between nitrogen and carbon atoms in side chains of histidines (Fig 3B), it would not be possible to distinguish nitrogen of ammonia from oxygen of water in the pore of AtTIP2;1 due to their similar electron density and expected low ammonia occupancy. To get a more detailed view of the substrate specificity in AtTIP2;1, we therefore employed MD simulations. Water permeation was seen at high frequency (pf ± SD) corresponding to approximately 25 ± 4 × 10−14 cm3 s−1 (S2 Fig), which is about four times as high as estimated for human AQP1. The high water permeation in AtTIP2;1 is consistent with its low free energy for water (S3 Fig). Notably, spontaneous ammonia permeation events were observed in unbiased simulations with a length of 400 ns (S1 Movie) and verified by umbrella sampling simulations yielding a free energy barrier of approximately 15 kJ/mol (Fig 6A) in line with a high ammonia permeability. Further analysis shows that desolvation effects are compensated for by several hydrogen bonding residues at the selectivity filter (Fig 6A–6C), substantially lowering the energetic barrier in this region, where it peaks for the water-specific HsAQP1 [25]. In contrast to a simple model membrane (S3 Fig), the tonoplast contains sterols [26], which increase the impermeability to polar molecules. Therefore, the ammonia permeability of AtTIP2;1 is compared to a cholesterol containing model membrane with a free energy barrier for ammonia of 20 kJ/mol (Fig 6A). Due to these differences in energy barriers, the permeability of AtTIP2;1 is an order of magnitude higher.


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)

MD simulations of AtTIP2;1.(A) The potential mean force (PMF) profiles for ammonia through AtTIP2;1 (red) and through a model membrane containing 20% cholesterol (green). In the lower part of the panel, the number of hydrogen bonds between ammonia and AtTIP2;1 are shown as function of position along the pore axis. Interactions with residues in the extended selectivity filter depicted in (C) are color-coded to resolve their contribution (H2P-His 63 (blue), LCP-His 131 (red), LEP-Gly 194 (green) and HEP-Arg 200 (brown)), and demonstrate hydrogen bonding to each of the four polar residues of the extended selectivity filter. (B) Snapshots of ammonia permeation. Cross section of AtTIP2;1 shown as grey surface and green cartoon of the backbone. Side chains of selected amino acid residues in the selectivity filter are displayed as sticks and color coded as in (C). (C) Close-up of an ammonia molecule at the center, forming hydrogen bonds to four residues (H2P-His 63, LCP-His 131, LEP-Gly 194, and HEP-Arg 200) of the selectivity filter. The hydrogen bonds are indicated by orange dashes and distances are given in Å. Ile 185 at position H5P of the selectivity filter, located in front of the visual plane, is not shown. The underlying data of panel A can be found in S1 Data.
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pbio.1002411.g006: MD simulations of AtTIP2;1.(A) The potential mean force (PMF) profiles for ammonia through AtTIP2;1 (red) and through a model membrane containing 20% cholesterol (green). In the lower part of the panel, the number of hydrogen bonds between ammonia and AtTIP2;1 are shown as function of position along the pore axis. Interactions with residues in the extended selectivity filter depicted in (C) are color-coded to resolve their contribution (H2P-His 63 (blue), LCP-His 131 (red), LEP-Gly 194 (green) and HEP-Arg 200 (brown)), and demonstrate hydrogen bonding to each of the four polar residues of the extended selectivity filter. (B) Snapshots of ammonia permeation. Cross section of AtTIP2;1 shown as grey surface and green cartoon of the backbone. Side chains of selected amino acid residues in the selectivity filter are displayed as sticks and color coded as in (C). (C) Close-up of an ammonia molecule at the center, forming hydrogen bonds to four residues (H2P-His 63, LCP-His 131, LEP-Gly 194, and HEP-Arg 200) of the selectivity filter. The hydrogen bonds are indicated by orange dashes and distances are given in Å. Ile 185 at position H5P of the selectivity filter, located in front of the visual plane, is not shown. The underlying data of panel A can be found in S1 Data.
Mentions: Although the high resolution structure of AtTIP2;1 allowed us to discriminate between nitrogen and carbon atoms in side chains of histidines (Fig 3B), it would not be possible to distinguish nitrogen of ammonia from oxygen of water in the pore of AtTIP2;1 due to their similar electron density and expected low ammonia occupancy. To get a more detailed view of the substrate specificity in AtTIP2;1, we therefore employed MD simulations. Water permeation was seen at high frequency (pf ± SD) corresponding to approximately 25 ± 4 × 10−14 cm3 s−1 (S2 Fig), which is about four times as high as estimated for human AQP1. The high water permeation in AtTIP2;1 is consistent with its low free energy for water (S3 Fig). Notably, spontaneous ammonia permeation events were observed in unbiased simulations with a length of 400 ns (S1 Movie) and verified by umbrella sampling simulations yielding a free energy barrier of approximately 15 kJ/mol (Fig 6A) in line with a high ammonia permeability. Further analysis shows that desolvation effects are compensated for by several hydrogen bonding residues at the selectivity filter (Fig 6A–6C), substantially lowering the energetic barrier in this region, where it peaks for the water-specific HsAQP1 [25]. In contrast to a simple model membrane (S3 Fig), the tonoplast contains sterols [26], which increase the impermeability to polar molecules. Therefore, the ammonia permeability of AtTIP2;1 is compared to a cholesterol containing model membrane with a free energy barrier for ammonia of 20 kJ/mol (Fig 6A). Due to these differences in energy barriers, the permeability of AtTIP2;1 is an order of magnitude higher.

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