title: Single amino acid substitutions in the selectivity filter render NbXIP1;1α aquaporin water permeable.
abstract: Aquaporins (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.
  
  
  
  Here, 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.
  
  
  
  The 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.
authors.affiliation: Center for Molecular Protein Science, Department of Biochemistry and Structural Biology, Lund University, Box 124, SE-221 00, Lund, Sweden.
authors.forename: Henry
authors.initials: H
authors.lastname: Ampah-Korsah
authors.affiliation: Center for Molecular Protein Science, Department of Biochemistry and Structural Biology, Lund University, Box 124, SE-221 00, Lund, Sweden.
authors.forename: Yonathan
authors.initials: Y
authors.lastname: Sonntag
authors.affiliation: Center for Molecular Protein Science, Department of Biochemistry and Structural Biology, Lund University, Box 124, SE-221 00, Lund, Sweden.
authors.forename: Angelica
authors.initials: A
authors.lastname: Engfors
authors.affiliation: Center for Molecular Protein Science, Department of Biochemistry and Structural Biology, Lund University, Box 124, SE-221 00, Lund, Sweden.
authors.forename: Andreas
authors.initials: A
authors.lastname: Kirscht
authors.affiliation: Center for Molecular Protein Science, Department of Biochemistry and Structural Biology, Lund University, Box 124, SE-221 00, Lund, Sweden.
authors.forename: Per
authors.initials: P
authors.lastname: Kjellbom
authors.affiliation: Center for Molecular Protein Science, Department of Biochemistry and Structural Biology, Lund University, Box 124, SE-221 00, Lund, Sweden. urban.johanson@biochemistry.lu.se.
authors.forename: Urban
authors.identifier: 0000-0001-8862-128X
authors.initials: U
authors.lastname: Johanson
mesh_terms: D019943:Amino Acid Substitution; D020346:Aquaporins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D010843:Pichia / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017384:Sequence Deletion; D040681:Structural Homology, Protein; D014026:Nicotiana / Q000737:chemistry*; D014867:Water / Q000737:chemistry / Q000378:metabolism
chemical_list: D020346:Aquaporins; D029681:Arabidopsis Proteins; D010940:Plant Proteins; C500688:TIP2;1 protein, Arabidopsis; D014867:Water
keywords: Boric acid; Homology modeling; MIPs; Major Intrinsic Proteins; Mutation; XIP