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.

The Eucalyptus Tonoplast Intrinsic Protein (TIP) Gene Subfamily: Genomic Organization, Structural Features, and Expression Profiles

“…To evaluate structural similarities and differences, theoretical models for EgTIPs representing each classical TIP group were constructed using a comparative modeling approach and the crystallography structure of AtTIP2.1 at 1.18Å resolution (Kirscht et al, 2016) as a template. The sequences shared 53–85% aa identity and 90–95% of coverage.…”

“…Further comparison of the EgTIP-deduced aa sequences with those of other plant TIPs revealed that TIP members of groups 1 and 2 have the most conserved selectivity filters, including the H residue located in loop C (LC) of the proposed extended ar/R filter (Kirscht et al, 2016) ( Figures 3D,E ). VvTIP1.3 from V. vinifera , that possesses an M residue instead of a V in the LE2 position, is the only exception.…”

“…Previous data have indicated that TIP subfamily members show the highest level of sequence divergence in the ar/R region among known plant aquaporins (Wallace and Roberts, 2004; Azad et al, 2012). Despite this, some residues are well conserved, particularly in the LE1 position, in which an A or G is present, and in the H 2 position, in which an H residue has been associated with water specificity (Sui et al, 2001; Kirscht et al, 2016). Remarkably, this conserved H residue is found in most identified EgTIPs, with the exception of EgTIP3.2 and EgTIP5.1.…”

“…These structures were predicted based on the alignment data generated by the program Phyre2 that uses homology detection methods to build 3D models (Kelley et al, 2015). Considering the quality of the structure, the crystallographic structure of the A. thaliana aquaporin TIP2.1 (AtTIP2.1) at 1.18 Å resolution (PDB ID: 5I32, Chain A) (Kirscht et al, 2016) was chosen as template. The program MODELLER 9v16 (Sali and Blundell, 1993; Marti-Renom et al, 2000) was used to generate the protein models and rank them according to the DOPE (Discrete Optimized Protein Energy) score.…”

“…Members of the Tonoplast Intrinsic Protein (TIP) subfamily are generally targeted to the vacuolar membrane and known to facilitate water transport across this subcellular compartment. Parallel to their role as water channels, TIP isoforms can also translocate glycerol, hydrogen peroxide (H 2 O 2 ) (reviewed in Maeshima, 2001) and urea (Liu et al, 2003), and enhance vacuolar membrane permeability to ammonia (Loqué et al, 2005; Kirscht et al, 2016). Specificity of substrate transport is mainly determined by four amino acid (aa) residues present at specific positions of the so-called aromatic/arginine (ar/R) selectivity filter, which acts as a size-exclusion barrier (Sui et al, 2001; Wu et al, 2009).…”