title: Metal binding to the N-terminal cytoplasmic domain of the PIB ATPase HMA4 is required for metal transport in Arabidopsis.
abstract: PIB ATPases are metal cation pumps that transport metals across membranes. These proteins possess N- and C-terminal cytoplasmic extensions that contain Cys- and His-rich high affinity metal binding domains, which may be involved in metal sensing, metal ion selectivity and/or in regulation of the pump activity. The PIB ATPase HMA4 (Heavy Metal ATPase 4) plays a central role in metal homeostasis in Arabidopsis thaliana and has a key function in zinc and cadmium hypertolerance and hyperaccumulation in the extremophile plant species Arabidopsis halleri. Here, we examined the function and structure of the N-terminal cytoplasmic metal-binding domain of HMA4. We mutagenized a conserved CCTSE metal-binding motif in the domain and assessed the impact of the mutations on protein function and localization in planta, on metal-binding properties in vitro and on protein structure by Nuclear Magnetic Resonance spectroscopy. The two Cys residues of the motif are essential for the function, but not for localization, of HMA4 in planta, whereas the Glu residue is important but not essential. These residues also determine zinc coordination and affinity. Zinc binding to the N-terminal domain is thus crucial for HMA4 protein function, whereas it is not required to maintain the protein structure. Altogether, combining in vivo and in vitro approaches in our study provides insights towards the molecular understanding of metal transport and specificity of metal P-type ATPases.
authors.affiliation: Department of Life Sciences, Center for Protein Engineering (CIP), University of Liège, 4000, Liège, Belgium.
authors.forename: Clémentine
authors.initials: C
authors.lastname: Laurent
authors.affiliation: Department of Life Sciences, Center for Protein Engineering (CIP), University of Liège, 4000, Liège, Belgium.
authors.forename: Gilles
authors.initials: G
authors.lastname: Lekeux
authors.affiliation: School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC, 3010, Australia.
authors.forename: Ashwinie A
authors.initials: AA
authors.lastname: Ukuwela
authors.affiliation: School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC, 3010, Australia.
authors.forename: Zhiguang
authors.initials: Z
authors.lastname: Xiao
authors.affiliation: Department of Life Sciences, Center for Protein Engineering (CIP), University of Liège, 4000, Liège, Belgium.
authors.forename: Jean-Benoit
authors.initials: JB
authors.lastname: Charlier
authors.affiliation: Laboratory of Plant and Microbial Ecology, Department of Biology, Ecology, Evolution, University of Liège, 4000, Liège, Belgium.
authors.forename: Bernard
authors.initials: B
authors.lastname: Bosman
authors.affiliation: Laboratory of Plant and Microbial Ecology, Department of Biology, Ecology, Evolution, University of Liège, 4000, Liège, Belgium.
authors.forename: Monique
authors.initials: M
authors.lastname: Carnol
authors.affiliation: Department of Life Sciences, Center for Protein Engineering (CIP), University of Liège, 4000, Liège, Belgium.|PhytoSYSTEMS, University of Liège, 4000, Liège, Belgium.
authors.forename: Patrick
authors.initials: P
authors.lastname: Motte
authors.affiliation: Chimie Biologique Structurale, Department of Chemistry, University of Liège, Liège, Belgium.
authors.forename: Christian
authors.initials: C
authors.lastname: Damblon
authors.affiliation: Department of Life Sciences, Center for Protein Engineering (CIP), University of Liège, 4000, Liège, Belgium.
authors.forename: Moreno
authors.initials: M
authors.lastname: Galleni
authors.affiliation: Department of Life Sciences, Center for Protein Engineering (CIP), University of Liège, 4000, Liège, Belgium. marc.hanikenne@ulg.ac.be.|PhytoSYSTEMS, University of Liège, 4000, Liège, Belgium. marc.hanikenne@ulg.ac.be.
authors.forename: Marc
authors.initials: M
authors.lastname: Hanikenne
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D002104:Cadmium / Q000378:metabolism; D002462:Cell Membrane; D003001:Cloning, Molecular; D018506:Gene Expression Regulation, Plant / Q000502:physiology*; D009682:Magnetic Resonance Spectroscopy; D008670:Metals / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D021381:Protein Transport; D015032:Zinc / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D008670:Metals; D002104:Cadmium; D000251:Adenosine Triphosphatases; C477864:HMA4 protein, Arabidopsis; D015032:Zinc
keywords: Arabidopsis; Metal P-type ATPase; Metal binding domain; Structure–function analysis; Zinc transport