Exploring Unorthodox Aquaporins : Characterization of NbXIPs & AtNIPs

University dissertation from Lund University, Faculty of Science, Department of Chemistry, Division of Biochemistry and Structural Biology

Abstract: AbstractAquaporins are membrane integral proteins that facilitate the transport of waterand/or other small neutral molecules across biological membranes in cells in allforms of life. Among the subfamilies of aquaporins in higher plants, the X IntrinsicProteins (XIPs) are the most recently discovered subfamily and the leastcharacterized. However, the aromatic/arginine (ar/R) selectivity filter and theproposed substrate specificity of XIPs resemble that of some NIPs aquaporinisoforms. The aim of the studies in this thesis was to functionally and structurallycharacterize Nicotiana benthamiana XIP1;1s and Arabidopsis thaliana NIP1;1 andNIP5;1 aquaporins.By using the Pichia pastoris expression system, NbXIP1;1s, AtNIP1;1 andAtNIP5;1 were successfully expressed. NbXIP1;1s and AtNIP5;1 increased thesensitivity of P. pastoris cells to boric acid. Furthermore, the N-terminally His-taggedsplice-variant NbXIP1;1α appeared more opened to boric acid than the C-terminallyHis-tagged protein when expressed in P. pastoris. In P. pastoris spheroplasts, differences in water and glycerol permeability of NbXIP1;1α and AtNIP5;1 were observed. AtNIP5;1 was permeable to water and glycerol but NbXIP1:1α was impermeable to both substrates. NbXIP1;1α and AtNIP5;1 proteins were purified by Ni-NTA affinity chromatography. While the yield obtained for NbXIP1;1α was adequate for both functional and initial crystallization studies, the initial yield for AtNIP5;1 was only sufficient for functional characterization. The current purification scheme for AtNIP5;1 needs to be optimized to obtain sufficient amounts of protein for a comprehensive crystallization study.In a stopped-flow spectrometric analysis, NbXIP1;1α was partially permeable toboric acid in proteoliposomes. Mass spectrometric analysis revealed that thepurified NbXIP1;1α protein was phosphorylated at five amino acid residues in theN-terminal region. Results from mutational studies suggested that NbXIP1;1α maybe regulated by its N-terminal domain. It was therefore proposed that NbXIP1;1α isgated by phosphorylation. By site-directed mutagenesis, it was shown that singleamino acid substitutions L79G in helix 1 and I102H in helix 2 were sufficient torender NbXIP1;1α water permeable. Water permeable NbXIP1;1α mutants offer ameans to further probe the functional properties of NbXIP1;1α. Homology modelsof wild-type and mutant NbXIP1;1α in conjunction with functional studies suggested that T246 is the residue at the helix 5 position in the ar/R filter of NbXIP1;1α. The models also revealed a previously unrecognized orientation and interaction of the conserved arginine in the ar/R filter, which could serve as a novel guide to tune the selectivity of aquaporins.