Crystal Structure of an Ammonia-Permeable Aquaporin

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.

Abstract

The structure of an ammonia channel from plants extends our understanding of substrate specificity in different types of aquaporins and reveals an intriguing side pore that raises new questions.

Author Summary

Ammonia is a central molecule in nitrogen metabolism. Aquaporins are integral membrane proteins that form channels that accelerate the passive permeation of small polar uncharged molecules, like water and ammonia, across lipid membranes of the cell. 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 permeability may, in the future, allow us to modulate the passage of ammonia and generate crops with higher nitrogen-use efficiency.