title: Identification of functionally important conserved trans-membrane residues of bacterial PIB -type ATPases.
abstract: Powered by ATP hydrolysis, P(IB) -ATPases drive the energetically uphill transport of transition metals. These high affinity pumps are essential for heavy metal detoxification and delivery of metal cofactors to specific cellular compartments. Amino acid sequence alignment of the trans-membrane (TM) helices of P(IB)-ATPases reveals a high degree of conservation, with ∼ 60-70 fully conserved positions. Of these conserved positions, 6-7 were previously identified to be important for transport. However, the functional importance of the majority of the conserved TM residues remains unclear. To investigate the role of conserved TM residues of P(IB)-ATPases we conducted an extensive mutagenesis study of a Zn(2+)/Cd(2+) P(IB)-ATPase from Rhizobium radiobacter (rrZntA) and seven other P(IB)-ATPases. Of the 38 conserved positions tested, 24 had small effects on metal tolerance. Fourteen mutations compromised in vivo metal tolerance and in vitro metal-stimulated ATPase activity. Based on structural modelling, the functionally important residues line a constricted 'channel', tightly surrounded by the residues that were found to be inconsequential for function. We tentatively propose that the distribution of the mutable and immutable residues marks a possible trans-membrane metal translocation pathway. In addition, by substituting six trans-membrane amino acids of rrZntA we changed the in vivo metal specificity of this pump from Zn(2+)/Cd(2+) to Ag(+).
authors.affiliation: Department of Microbiology, The Bruce and Ruth Rappaport Faculty of Medicine, The Technion-Israel Institute of Technology, Haifa, Israel.
authors.forename: Daniel
authors.initials: D
authors.lastname: Zhitnitsky
authors.forename: Oded
authors.initials: O
authors.lastname: Lewinson
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008670:Metals / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013379:Substrate Specificity; D028561:Transition Elements / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D008670:Metals; D028561:Transition Elements; D000251:Adenosine Triphosphatases