Single amino acid substitutions in the selectivity filter render NbXIP1;1α aquaporin water permeable

Abstract

BackgroundAquaporins (AQPs) are integral membrane proteins that facilitate transport of water and/or other small neutral solutes across membranes in all forms of life. The X Intrinsic Proteins (XIPs) are the most recently recognized and the least characterized aquaporin subfamily in higher plants. XIP1s have been shown to be impermeable to water but permeable to boric acid, glycerol, hydrogen peroxide and urea. However, uncertainty regarding the determinants for selectivity and lack of an activity that is easy to quantify have hindered functional investigations. In an effort to resolve these issues, we set out to introduce water permeability in Nicotiana benthamiana XIP1;1α (NbXIP1;1α), by exchanging amino acid residues of predicted alternative aromatic/arginine (ar/R) selectivity filters of NbXIP1;1α for residues constituting the water permeable ar/R selectivity filter of AtTIP2;1.ResultsHere, we present functional results regarding the amino acid substitutions in the putative filters as well as deletions in loops C and D of NbXIP1;1α. In addition, homology models were created based on the high resolution X-ray structure of AtTIP2;1 to rationalize the functional properties of wild-type and mutant NbXIP1;1α. Our results favour Thr 246 rather than Val 242 as the residue at the helix 5 position in the ar/R filter of NbXIP1;1α and indicate that the pore is not occluded by the loops when heterologously expressed in Pichia pastoris. Moreover, our results show that a single amino acid substitution in helix 1 (L79G) or in helix 2 (I102H) is sufficient to render NbXIP1;1α water permeable. Most of the functional results can be rationalized from the models based on a combination of aperture and hydrophobicity of the ar/R filter.ConclusionThe water permeable NbXIP1;1α mutants imply that the heterologously expressed proteins are correctly folded and offer means to explore the structural and functional properties of NbXIP1;1α. Our results support that Thr 246 is part of the ar/R filter. Furthermore, we suggest that a salt bridge to an acidic residue in helix 1, conserved among the XIPs in clade B, directs the orientation of the arginine in the ar/R selectivity filter and provides a novel approach to tune the selectivity of AQPs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-017-1009-3) contains supplementary material, which is available to authorized users.

Overexpression of X Intrinsic Protein 1;1 in Nicotiana tabacum and Arabidopsis reduces boron allocation to shoot sink tissues

“…The first molecular characterization of plant XIPs demonstrated that NtXIP1;1 from tobacco is localized in the plasma membrane and that its promoter is widely active throughout the plant, both in roots and shoots (Bienert et al., ). In N. tabacum and Nicotiana benthamiana , the transcript of XIP1;1 is spliced in two distinct variants, XIP1;1 α and XIP1;1 β differing in only one amino acid in length (Ampah‐Korsah et al., , ; Bienert et al., ). Toxicity growth assays in the heterologous expression systems Saccharomyces cerevisiae and Pichia pastoris , respectively, suggest that NtXIP1;1α, NtXIP1;1β and NbXIP1;1α function as boric acid channels (Ampah‐Korsah et al., ; Bienert et al., ).…”

Insights into the Selectivity Mechanisms of Grapevine NIP Aquaporins

“…The critical role of the ar/R region in many plant MIPs subfamilies, like PIPs [ 19 ], TIPs [ 20 ], and XIPs [ 21 ], has been elucidated by the substitution of key amino acids (reviewed by Reference [ 22 ]). However, only a few studies have addressed NIPs, despite being considered as crucial channels for transport of various beneficial (like B and Si), as well as toxic (like As), metalloids in plants [ 23 , 24 , 25 , 26 ].…”