Exploring Unorthodox Aquaporins : Characterization of NbXIPs & AtNIPs

Abstract

Abstract
Aquaporins are membrane integral proteins that facilitate the transport of water
and/or other small neutral molecules across biological membranes in cells in all
forms of life. Among the subfamilies of aquaporins in higher plants, the X Intrinsic
Proteins (XIPs) are the most recently discovered subfamily and the least
characterized. However, the aromatic/arginine (ar/R) selectivity filter and the
proposed substrate specificity of XIPs resemble that of some NIPs aquaporin
isoforms. The aim of the studies in this thesis was to functionally and structurally
characterize Nicotiana benthamiana XIP1;1s and Arabidopsis thaliana NIP1;1 and
NIP5;1 aquaporins.
By using the Pichia pastoris expression system, NbXIP1;1s, AtNIP1;1 and
AtNIP5;1 were successfully expressed. NbXIP1;1s and AtNIP5;1 increased the
sensitivity of P. pastoris cells to boric acid. Furthermore, the N-terminally His-tagged
splice-variant NbXIP1;1α appeared more opened to boric acid than the C-terminally
His-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 to
boric acid in proteoliposomes. Mass spectrometric analysis revealed that the
purified NbXIP1;1α protein was phosphorylated at five amino acid residues in the
N-terminal region. Results from mutational studies suggested that NbXIP1;1α may
be regulated by its N-terminal domain. It was therefore proposed that NbXIP1;1α is
gated by phosphorylation. By site-directed mutagenesis, it was shown that single
amino acid substitutions L79G in helix 1 and I102H in helix 2 were sufficient to
render NbXIP1;1α water permeable. Water permeable NbXIP1;1α mutants offer a
means to further probe the functional properties of NbXIP1;1α. Homology models
of 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.

Abstract (Swedish)

Popular science summary

Water is very important for life. In order to survive every organism needs to control the amount of water in its cells. One of the main ways the cell regulates its water content is through aquaporins. Aquaporins are membrane proteins that allow water and/or small neutral molecules to enter or exit the cell. Aquaporins form a large family and are present in all life forms, however, plants have more aquaporins than other organisms. Aquaporins are also known as Major Intrinsic Proteins (MIPs). Members belonging to the aquaporin family have the same overall three dimensional (3D) structure, however, some aquaporins have different selectivity filters. Orthodox or classical aquaporins are aquaporins that are permeable to water

only as the name aquaporin suggests. Unorthodox aquaporins are aquaporins that have selectivity filters that allow other small neutral molecules to pass.

In 2008, new members of the aquaporin family were recognized in the early land plants, i.e. mosses. Since the function of these new aquaporin members was not known, they were called X intrinsic proteins (XIPs), X standing for unknown function. However, the selectivity filter of XIPs resemble that of some previously

known plant aquaporins called Nodulin 26-like Intrinsic Proteins (NIPs). Different XIPs have been reported to facilitate the transport of boric acid, hydrogen peroxide, urea, glycerol but not water. However, the physiological function of XIPs is still unknown. Therefore, the purpose of the projects in this thesis was to increase the knowledge about Nicotiana benthamiana XIPs (NbXIPs) and Arabidopsis thaliana NIPs (AtNIPs) aquaporins.

The yeast, Pichia pastoris, has previously been used to produce and study many proteins including aquaporins. P. pastoris was therefore used to produce NbXIP1;1s, AtNIP1;1 and AtNIP5;1 aquaporins. Since the production of NbXIP1:1s and AtNIP5;1 was better than that of AtNIP1;1 in the yeast, NbXIP1;1s and

AtNIP5;1 were chosen for further studies. Both NbXIP1;1s and AtNIP5;1 facilitated the transport of boric acid into intact yeast cells which retarded the growth of the cells since boric acid is toxic to yeast. On the contrary, boric acid is beneficial to plants, as plants need boron for proper growth and yield. In P. pastoris cells with weakened cell walls, the splice-variant NbXIP1;1α was not permeable to water or glycerol, while AtNIP5;1 was permeable to both molecules. In order to study NbXIP1;1α and AtNIP5;1 proteins in a controlled environment away from interfering effects of other proteins, NbXIP1;1α and AtNIP5;1 were isolated and purified from the yeast cells. Though the amount of purified AtNIP5;1 obtained after the initial purification trial was enough for functional studies, it was not enough for crystallization studies aimed at solving the 3D structure of AtNIP5;1. Therefore, the initial purification protocol for AtNIP5;1 will be improved to generate large amounts of pure AtNIP5;1 protein for crystallization studies. A 3D structure of AtNIP5;1, together with functional studies, will help clarify how the structure of AtNIP5;1 affects its function. However, the yield of purified NbXIP1;1α obtained after purification was sufficient for both functional and crystallization studies.

The transport capabilities of purified aquaporins are usually studied by inserting aquaporins into lipid vesicles and measuring the shrinking or swelling of the vesicles in response to rapid changes in sugar or salt concentration. A rapid change in boric acid concentration revealed that lipid vesicles with inserted purified

NbXIP1;1α protein were two-fold more permeable to boric acid as compared to empty lipid vesicles. An examination of the NbXIP1;1α protein revealed that the purified NbXIP1;1α protein was partially modified with phosphates. Five amino acid residues in the N-terminal part of the protein had phosphate groups attached. Phosphate modification of aquaporins usually controls aquaporins in two ways; by serving as a signal to transport the aquaporin to a specific membrane location in the cell or by causing a structural change in the aquaporin that allows the pore to open. When the N-terminal part of NbXIP1;1α with the phosphate groups was cut from the protein, the protein became more permeable to boric acid in intact yeast cells. This suggested that the N-terminal part of NbXIP1;1α regulates its function, e.g. permeability. In an attempt to understand how the selectivity filter of NbXIP1;1s works, the water-impermeable filter of NbXIP1;1α was exchanged for the water permeable filter of AtTIP2;1, a close relative of XIPs.

The selectivity filter of aquaporins is made up of five amino acid residues that determine which molecules can pass through the pore of the aquaporin. However, a substitution of only one of the selectivity filter residues made the NbXIP1;1α protein water permeable. Interestingly, a substitution of another residue outside the selectivity filter also made NbXIP1;1α water permeable. The water permeable NbXIP1;1α constructs provide the possibility to explore the functional properties of NbXIP1;1s in great detail. AtNIP5;1 has been shown to be one of the proteins responsible for boric acid transport into plant cells. Since the selectivity filter and the choice of substrates of NbXIP1;1s are similar to that of AtNIP5;1, a possible

physiological function of NbXIP1;1s could be that they facilitate the transport of boric acid into distinct plant cells.

• author: Ampah-Korsah, Henry
• supervisor: Per Kjellbom; Urban Johanson
• opponent: Dr. Bienert, Gerd Patrick, Leibniz Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany
• organization: Biochemistry and Structural Biology
• publishing date: 2016
• type: Thesis
• publication status: published
• subject: Biochemistry and Molecular Biology; Structural Biology
• keywords: Aquaporins, XIPs, NIPs, Phosphorylation, Pichia pastoris, Overexpression, boric acid, membrane proteins
• pages: 184 pages
• defense location: Center for chemistry and chemical engineering, lecture hall B, Naturvetarvägen 14, Lund
• defense date: 2016-10-28 09:15:00
• ISBN: 978-91-7422-474-0
• ISBN: 978-91-7422-475-7
• language: English
• LU publication?: yes
• id: 110a0099-a085-49fc-a31c-6ca0bef305cd
• date added to LUP: 2016-09-29 17:06:14
• date last changed: 2018-11-21 21:26:15