Crystal Structure of an Ammonia-Permeable Aquaporin

PLoS Biology, Mar 2016

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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Crystal Structure of an Ammonia-Permeable Aquaporin

March Crystal Structure of an Ammonia-Permeable Aquaporin Andreas Kirscht 0 1 2 3 Shreyas S. Kaptan 0 1 2 3 Gerd Patrick Bienert 0 1 2 3 François Chaumont 0 1 2 3 Poul Nissen 0 1 2 3 Bert L. de Groot 0 1 2 3 Per Kjellbom 0 1 2 3 Pontus Gourdon 0 1 2 3 Urban Johanson 0 1 2 3 Author Summary 0 1 2 3 0 1 Department of Biochemistry and Structural Biology, Center for Molecular Protein Science, Lund University, Lund, Sweden, 2 The Max Planck Institute for Biophysical Chemistry, Computational Biomolecular Dynamics Group , Göttingen, Germany , 3 Institut des Sciences de la Vie, Université catholique de Louvain , Louvain-la-Neuve , Belgium , 4 IPK-Leibniz Institute of Plant Genetics and Crop Plant Research Department of Physiology and Cell Biology, Gatersleben, Germany, 5 Danish Research Institute of Translational Neuroscience-DANDRITE, Nordic-EMBL Partnership for Molecular Medicine, Department of Molecular Biology and Genetics, Aarhus University , Aarhus C , Denmark 1 Funding: Financial support from the Swedish Research Council (VR) UJ, PK; Formas, the Research School of Pharmaceutical Sciences (FLÄK) PK; the Belgian National Fund for Scientific Research (FNRS), the Interuniversity Attraction Poles Programme-Belgian Science Policy, and the “Communauté française de Belgique-Actions de Recherches Concertées” are gratefully 2 Academic Editor: Raimund Dutzler, University of Zurich , SWITZERLAND 3 Current address: Department of Biomedical Sciences, University of Copenhagen , Blegdamsvej 3B, DK- 2200 Copenhagen, Denmark , and Department of Experimental Medical Science, Lund University , Sölvegatan 19, SE-221 84 Lund , Sweden 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. - acknowledged FC; GPB was an FNRS Postdoctoral Researcher and is currently supported by an Emmy Noether grant 1668/1-1 from the Deutsche Forschungsgemeinschaft. PN was supported by an advanced research grant (Biomemos) of the European Research Council. Access to synchrotron sources was supported by the Danscatt program of the Danish Council of Independent Research, and by BioStruct-X contract 860. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: AQP, aquaporin; MD, molecular dynamics; NPA, Asn-Pro-Ala; OG, n-octyl-β-Dglucoside; PIP, plasma membrane intrinsic protein; PMF, potential mean force; POPC, 1-Palmitoyl-2oleoylphosphatidylcholine; TIP, Tonoplast Intrinsic Protein. Structural information of ammonia-permeable aquaporins has been lacking. Here, we report a high-resolution structure of the ammonia-permeable aquaporin AtTIP2;1 and explore it by functional assays of mutants and by molecular dynamics simulations. Our data uncover unexpected features of the substrate selectivity filter, including a conserved arginine in a new orientation that is stabilized by interactions to a histidine that is linked to ammonia specificity. An additional histidine in a different part of AtTIP2;1 fortifies the position of the arginine and interacts directly with the substrate in the channel. This histidine is therefore included in an extended selectivity filter, which should prompt a reinterpretation of the determinants of specificity in all types of aquaporins. We speculate that an intriguing water-filled side pore, next to the substrate-binding histidine, participates in deprotonating ammonium ions, which could increase the net permeation of ammonia. Understanding the principles of ammonia permeabi (...truncated)


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Andreas Kirscht, Shreyas S. Kaptan, Gerd Patrick Bienert, François Chaumont, Poul Nissen, Bert L. de Groot, Per Kjellbom, Pontus Gourdon, Urban Johanson. Crystal Structure of an Ammonia-Permeable Aquaporin, PLoS Biology, 2016, Volume 14, Issue 3, DOI: 10.1371/journal.pbio.1002411