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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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Growth complementation of ammonium uptake-defective yeast strain by mutants of AtTIP2;1.The 31019b yeast strain (Δmep1–3) was transformed with the empty vector pYeDP60u or with pYeDP60u-carrying cDNA encoding the positive controls HsAQP8 or HsAQP1, or AtTIP2;1 or its mutants. The five amino acid residues of the extended selectivity filter in each construct are indicated in one letter code to the left in the order H2P, LCP, H5P, LEP, and HEP, showing substitutions in bold. Complementation and failure to complement are indicated by + and −, respectively. Transformants were spotted at an OD600 of 1 (right column) and 0.01 (left column) on plates containing 0.2% proline or the indicated concentrations of ammonium as a sole nitrogen source and growth was recorded after 13 d at 28°C. Each panel showing growth at a specific concentration is compiled by individual pictures of each spot taken from a distinct growth condition. All single pictures were treated in the same manner.
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pbio.1002411.g004: Growth complementation of ammonium uptake-defective yeast strain by mutants of AtTIP2;1.The 31019b yeast strain (Δmep1–3) was transformed with the empty vector pYeDP60u or with pYeDP60u-carrying cDNA encoding the positive controls HsAQP8 or HsAQP1, or AtTIP2;1 or its mutants. The five amino acid residues of the extended selectivity filter in each construct are indicated in one letter code to the left in the order H2P, LCP, H5P, LEP, and HEP, showing substitutions in bold. Complementation and failure to complement are indicated by + and −, respectively. Transformants were spotted at an OD600 of 1 (right column) and 0.01 (left column) on plates containing 0.2% proline or the indicated concentrations of ammonium as a sole nitrogen source and growth was recorded after 13 d at 28°C. Each panel showing growth at a specific concentration is compiled by individual pictures of each spot taken from a distinct growth condition. All single pictures were treated in the same manner.

Mentions: To investigate the contribution of the amino acid residues in the extended selectivity filter to substrate specificity, we analyzed the permeability of the water-specific human AQP1 (HsAQP1) and AtTIP2;1 using in vivo and in vitro assays. Both these channels have the conserved arginine at position HEP of the selectivity filter but, as established by our structure, the spatial location of its side chain relative to the pore differs. Expectedly, substituting all four deviating residues of the extended selectivity filter in a quadruple mutant of AtTIP2;1 to the corresponding residues of HsAQP1 abolished complementation in a yeast growth assay probing for ammonia permeability (Fig 4). Single point mutations at each of the four positions specify that all of the individual substitutions except the exchange of histidine for phenylalanine at H2P are compatible with ammonia permeability in AtTIP2;1. The incompatibility of the phenylalanine may be explained by its different electrostatics and slightly larger size, which would be in conflict with the orientation of the arginine at position HEP in the selectivity filter of AtTIP2;1. The spatial orientation of the arginine at position HEP in AtTIP2;1 is constrained by loop C and the histidine at position LCP, hence the introduced phenylalanine at H2P is likely to adopt an alternative position where it occludes the pore. The compatibility of the three other single mutations may at first appearance be explained by their polar nature, allowing formation of hydrogen bonds to ammonia. However, as mentioned above, at position LEP, the hydrogen bond to the substrate is offered by the backbone carbonyl rather than the side chain. Interestingly, a double mutation in helix 5 (position H5P) and loop E (position LEP) of the selectivity filter failed to demonstrate ammonia permeability in the growth assay. This may indicate that a small and flexible residue at position LEP of AtTIP2;1 is required to allow its carbonyl to interact with the substrate in the presence of a histidine at position H5P.


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)

Growth complementation of ammonium uptake-defective yeast strain by mutants of AtTIP2;1.The 31019b yeast strain (Δmep1–3) was transformed with the empty vector pYeDP60u or with pYeDP60u-carrying cDNA encoding the positive controls HsAQP8 or HsAQP1, or AtTIP2;1 or its mutants. The five amino acid residues of the extended selectivity filter in each construct are indicated in one letter code to the left in the order H2P, LCP, H5P, LEP, and HEP, showing substitutions in bold. Complementation and failure to complement are indicated by + and −, respectively. Transformants were spotted at an OD600 of 1 (right column) and 0.01 (left column) on plates containing 0.2% proline or the indicated concentrations of ammonium as a sole nitrogen source and growth was recorded after 13 d at 28°C. Each panel showing growth at a specific concentration is compiled by individual pictures of each spot taken from a distinct growth condition. All single pictures were treated in the same manner.
© Copyright Policy
Related In: Results  -  Collection

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

pbio.1002411.g004: Growth complementation of ammonium uptake-defective yeast strain by mutants of AtTIP2;1.The 31019b yeast strain (Δmep1–3) was transformed with the empty vector pYeDP60u or with pYeDP60u-carrying cDNA encoding the positive controls HsAQP8 or HsAQP1, or AtTIP2;1 or its mutants. The five amino acid residues of the extended selectivity filter in each construct are indicated in one letter code to the left in the order H2P, LCP, H5P, LEP, and HEP, showing substitutions in bold. Complementation and failure to complement are indicated by + and −, respectively. Transformants were spotted at an OD600 of 1 (right column) and 0.01 (left column) on plates containing 0.2% proline or the indicated concentrations of ammonium as a sole nitrogen source and growth was recorded after 13 d at 28°C. Each panel showing growth at a specific concentration is compiled by individual pictures of each spot taken from a distinct growth condition. All single pictures were treated in the same manner.
Mentions: To investigate the contribution of the amino acid residues in the extended selectivity filter to substrate specificity, we analyzed the permeability of the water-specific human AQP1 (HsAQP1) and AtTIP2;1 using in vivo and in vitro assays. Both these channels have the conserved arginine at position HEP of the selectivity filter but, as established by our structure, the spatial location of its side chain relative to the pore differs. Expectedly, substituting all four deviating residues of the extended selectivity filter in a quadruple mutant of AtTIP2;1 to the corresponding residues of HsAQP1 abolished complementation in a yeast growth assay probing for ammonia permeability (Fig 4). Single point mutations at each of the four positions specify that all of the individual substitutions except the exchange of histidine for phenylalanine at H2P are compatible with ammonia permeability in AtTIP2;1. The incompatibility of the phenylalanine may be explained by its different electrostatics and slightly larger size, which would be in conflict with the orientation of the arginine at position HEP in the selectivity filter of AtTIP2;1. The spatial orientation of the arginine at position HEP in AtTIP2;1 is constrained by loop C and the histidine at position LCP, hence the introduced phenylalanine at H2P is likely to adopt an alternative position where it occludes the pore. The compatibility of the three other single mutations may at first appearance be explained by their polar nature, allowing formation of hydrogen bonds to ammonia. However, as mentioned above, at position LEP, the hydrogen bond to the substrate is offered by the backbone carbonyl rather than the side chain. Interestingly, a double mutation in helix 5 (position H5P) and loop E (position LEP) of the selectivity filter failed to demonstrate ammonia permeability in the growth assay. This may indicate that a small and flexible residue at position LEP of AtTIP2;1 is required to allow its carbonyl to interact with the substrate in the presence of a histidine at position H5P.

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