Pore Selectivity and Gating of Arabidopsis Nodulin 26 Intrinsic Proteins and Roles in Boric acid Transport in Reproductive Growth

Plant nodulin-26 intrinsic proteins (NIPs) are members of the aquaporin superfamily that serve as multifunctional channels of uncharged metabolites and water. They share the same canonical hourglass fold as the aquaporin family. The aromatic arginine (ar/R) selectivity filter controls transport selectivity based on size, hydrophobicity, and hydrogen bonding with substrates. In Arabidopsis thaliana, NIP II subclass proteins contain a conserved ar/R “pore signature” that is composed of Alanine at the helix 2 position (H2), Valine/Isoleucine at the helix 5 position (H5), and an Alanine (LE1) and an invariant Arginine (LE2) at the two loop E positions. In this study, we focused on the structure and function of a Arabidopsis thaliana NIP II protein, NIP7;1. Instead of being constitutive boric acid channel, NIP7;1 is a gated boric acid channel in Arabidopsis. We identified a conserved tyrosine residue (Tyr81) of NIP7;1 stabilizes a closed pore conformation through interaction with the canonical Arg220. Cysteine substitution results in opening of the pore that acquires a robust, transport activity for boric acid. Phenylalanine substitution also opens the channel, supporting the prediction from MD simulations that hydrogen bond interaction between the Tyr81 phenol group and the Arg220 may contribute to the stabilization of a closed pore state. Biological function studies indicate that NIP7;1 is an anther specific boric acid channel that plays a crucial role in the transport of boric acid in developing microspores. Both NIP7;1 promoter-GUS and NIP7;1-YFP fusion protein expression pattern suggested that NIP7;1 is predominantly expressed in stage 9 to 11 anthers, developing pollen and surrounding tapetum cells. T-DNA insertion lines of NIP7;1 showed moderate male sterility and abnormal pollen development under limited boron condition, especially the defect of pollen cell wall structure. Selectivity filter studies indicate that H2 position residue is the signature to differentiate the NIP I and NIP II subgroup proteins. Site-direct mutagenesis studies suggested that changing the H2 residue of NIP I (Tryptophan) to NIP II (Alanine) increases the boric acid while decreases water permeability. Molecular dynamics simulations and umbrella sampling identified a potential water selective residue: Arg220 whose dynamics could be fined tuned by the H2 position residue.