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Identification of functionally important conserved trans-membrane residues of bacterial PIB -type ATPases.

Zhitnitsky D, Lewinson O - Mol. Microbiol. (2014)

Bottom Line: Of the 38 conserved positions tested, 24 had small effects on metal tolerance.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.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(+).

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, The Bruce and Ruth Rappaport Faculty of Medicine, The Technion-Israel Institute of Technology, Haifa, Israel.

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An rrZntA mutant that confers Ag+ tolerance. Cells were transformed with an empty plasmid (circles), plasmid encoding wild type rrZntA (open squares), or plasmid encoding the rrZntA mutant I347M /L860Y/K861N/D882P/V889M/A893S (crosses). Cultures were grown in the absence or presence of the indicated AgNO3 concentrations. Error bars (shown unless smaller than icons) represent standard deviations of six repeats. Also shown are P-values derived by a Student's t-test.
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fig08: An rrZntA mutant that confers Ag+ tolerance. Cells were transformed with an empty plasmid (circles), plasmid encoding wild type rrZntA (open squares), or plasmid encoding the rrZntA mutant I347M /L860Y/K861N/D882P/V889M/A893S (crosses). Cultures were grown in the absence or presence of the indicated AgNO3 concentrations. Error bars (shown unless smaller than icons) represent standard deviations of six repeats. Also shown are P-values derived by a Student's t-test.

Mentions: rrZntA shares ∼36% overall amino acid sequence identity with pumps of Cu+/Ag+, yet the conservation is mostly limited to the soluble catalytic N, A and P domains. The sequence identity of the TMs is much lower (∼10%). We nevertheless attempted to identify a minimal set of trans-membrane amino acid substitutions that will change the selectivity of rrZntA from Zn2+/Cd2+ to Cu+/Ag+. We first introduced four residues that have been reported to be important for monovalent metal recognition: the Tyr and Asn of TM7, and the Met and Ser of TM8 (Mandal et al., 2004; González-Guerrero et al., 2008). However, the resulting mutant (L860Y/K861N/V889M/A893S) had no appreciable activity with either Cu+ or Ag+ as judged by metal tolerance experiments (Table S1). We made an additional substitution that introduced a Met residue in TM3. Based on the structure of lpCopA, this Met residue was proposed to be part of the ion entry site (Gourdon et al., 2011). Nevertheless, this variant (I347M/L860Y/K861N/V889M/A893S) also did not have any appreciable activity. We then took out this Met substitution and instead substituted the essential Asp882 (TM8) of rrZntA with a Pro residue. Although this proline has no known function, it is invariably conserved in pumps of Cu+/Ag+ (Fig. 1). Similar to what we observed with the previous constructs, we did not detect any appreciable activity with this one (L860Y/K861N/D882P/V889M/A893S) either. In a final attempt, we combined the two latter substitutions and generated a construct with a total of six substitutions: I347M /L860Y/K861N/D882P/V889M/A893S. Of the six mutations present in this mutant three are in residues that we found to be important for Zn2+/Cd2+ tolerance and metal-stimulated ATPase activity (L860, K861, D882, Table 1). Not surprisingly, this mutant lost all ability to confer Zn2+/Cd2+ tolerance (not shown). On the other hand, the mutant conferred robust Ag+ tolerance: in the presence of 4.5 μM AgNO3 the growth of this mutant was sevenfold greater than that of wild-type rrZntA, and ∼30-fold greater than that of cells transformed with a control plasmid (Fig. 8). Surprisingly, the altered metal specificity did not extend to Cu+, towards which no tolerance was observed (Table S1).


Identification of functionally important conserved trans-membrane residues of bacterial PIB -type ATPases.

Zhitnitsky D, Lewinson O - Mol. Microbiol. (2014)

An rrZntA mutant that confers Ag+ tolerance. Cells were transformed with an empty plasmid (circles), plasmid encoding wild type rrZntA (open squares), or plasmid encoding the rrZntA mutant I347M /L860Y/K861N/D882P/V889M/A893S (crosses). Cultures were grown in the absence or presence of the indicated AgNO3 concentrations. Error bars (shown unless smaller than icons) represent standard deviations of six repeats. Also shown are P-values derived by a Student's t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4285229&req=5

fig08: An rrZntA mutant that confers Ag+ tolerance. Cells were transformed with an empty plasmid (circles), plasmid encoding wild type rrZntA (open squares), or plasmid encoding the rrZntA mutant I347M /L860Y/K861N/D882P/V889M/A893S (crosses). Cultures were grown in the absence or presence of the indicated AgNO3 concentrations. Error bars (shown unless smaller than icons) represent standard deviations of six repeats. Also shown are P-values derived by a Student's t-test.
Mentions: rrZntA shares ∼36% overall amino acid sequence identity with pumps of Cu+/Ag+, yet the conservation is mostly limited to the soluble catalytic N, A and P domains. The sequence identity of the TMs is much lower (∼10%). We nevertheless attempted to identify a minimal set of trans-membrane amino acid substitutions that will change the selectivity of rrZntA from Zn2+/Cd2+ to Cu+/Ag+. We first introduced four residues that have been reported to be important for monovalent metal recognition: the Tyr and Asn of TM7, and the Met and Ser of TM8 (Mandal et al., 2004; González-Guerrero et al., 2008). However, the resulting mutant (L860Y/K861N/V889M/A893S) had no appreciable activity with either Cu+ or Ag+ as judged by metal tolerance experiments (Table S1). We made an additional substitution that introduced a Met residue in TM3. Based on the structure of lpCopA, this Met residue was proposed to be part of the ion entry site (Gourdon et al., 2011). Nevertheless, this variant (I347M/L860Y/K861N/V889M/A893S) also did not have any appreciable activity. We then took out this Met substitution and instead substituted the essential Asp882 (TM8) of rrZntA with a Pro residue. Although this proline has no known function, it is invariably conserved in pumps of Cu+/Ag+ (Fig. 1). Similar to what we observed with the previous constructs, we did not detect any appreciable activity with this one (L860Y/K861N/D882P/V889M/A893S) either. In a final attempt, we combined the two latter substitutions and generated a construct with a total of six substitutions: I347M /L860Y/K861N/D882P/V889M/A893S. Of the six mutations present in this mutant three are in residues that we found to be important for Zn2+/Cd2+ tolerance and metal-stimulated ATPase activity (L860, K861, D882, Table 1). Not surprisingly, this mutant lost all ability to confer Zn2+/Cd2+ tolerance (not shown). On the other hand, the mutant conferred robust Ag+ tolerance: in the presence of 4.5 μM AgNO3 the growth of this mutant was sevenfold greater than that of wild-type rrZntA, and ∼30-fold greater than that of cells transformed with a control plasmid (Fig. 8). Surprisingly, the altered metal specificity did not extend to Cu+, towards which no tolerance was observed (Table S1).

Bottom Line: Of the 38 conserved positions tested, 24 had small effects on metal tolerance.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.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(+).

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, The Bruce and Ruth Rappaport Faculty of Medicine, The Technion-Israel Institute of Technology, Haifa, Israel.

Show MeSH
Related in: MedlinePlus