title: Structural and functional analyses of PpENA1 provide insights into cation binding by type IID P-type ATPases in lower plants and fungi.
abstract: PpENA1 is a membrane-spanning transporter from the moss Physcomitrella patens, and is the first type IID P-type ATPase to be reported in the plant kingdom. In Physcomitrella, PpENA1 is essential for normal growth under moderate salt stress, while in yeast, type IID ATPases provide a vital efflux mechanism for cells under high salt conditions by selectively transporting Na+ or K+ across the plasma membrane. To investigate the structural basis for cation-binding within the type IID ATPase subfamily, we used homology modeling to identify a highly conserved cation-binding pocket between membrane helix (MH) 4 and MH 6 of the membrane-spanning pore of PpENA1. Mutation of specific charged and polar residues on MHs 4-6 resulted in a decrease or loss of protein activity as measured by complementation assays in yeast. The E298S mutation on MH 4 of PpENA1 had the most significant effect on activity despite the presence of a serine at this position in fungal type IID ATPases. Activity was partially restored in an inactivated PpENA1 mutant by the insertion of two additional serine residues on MH 4 and one on MH 6 based on the presence of these residues in fungal type IID ATPases. Our results suggest that the residues responsible for cation-binding in PpENA1 are distinct from those in fungal type IID ATPases, and that a fungal-type cation binding site can be successfully engineered into the moss protein.
authors.affiliation: Australian Centre for Plant Functional Genomics, University of Adelaide, Waite Campus, Glen Osmond, South Australia 5064, Australia. dpd@life.ku.dk
authors.forename: Damian P
authors.initials: DP
authors.lastname: Drew
authors.forename: Maria
authors.initials: M
authors.lastname: Hrmova
authors.forename: Christina
authors.initials: C
authors.lastname: Lunde
authors.forename: Andrew K
authors.initials: AK
authors.lastname: Jacobs
authors.forename: Mark
authors.initials: M
authors.lastname: Tester
authors.forename: Geoffrey B
authors.initials: GB
authors.lastname: Fincher
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D019068:Bryopsida / Q000201:enzymology*; D002412:Cations / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005658:Fungi / Q000201:enzymology; D005816:Genetic Complementation Test; D015151:Immunoblotting; D017136:Ion Transport; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D002412:Cations; D005656:Fungal Proteins; D010940:Plant Proteins; C559659:PpENA1 protein, Physcomitrella patens; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium