J Bacteriol. 2016 Jan 19. pii: JB.00975-15. [Epub ahead of print]

Functional determinants of metal ion transport and selectivity in paralogous cation diffusion facilitator transporters CzcD and MntE in Streptococcus pneumoniae.

Martin JE1, Giedroc DP2.

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Cation diffusion facilitators (CDFs) are a large family of divalent metal transporters that collectively possess broad metal specificity and contribute to intracellular metal homeostasis and virulence in bacterial pathogens.Streptococcus pneumoniae expresses two homologous CDF efflux transporters, MntE and CzcD. Cells lacking mntE or czcD are sensitive to manganese (Mn) or zinc (Zn) toxicity and specifically accumulate Mn or Zn, respectively, thus suggesting that MntE selectively transports Mn, while CzcD transports Zn. Here, we probe the origin of this metal specificity using a phenotypic growth analysis of pneumococcal variants. Structural homology to E. coli YiiP predicts that both MntE and CzcD are dimeric, and each protomer harbors four pairs of conserved metal-binding sites, termed the A-site, B-site, and C1/C2 binuclear site. We find that single amino acid mutations within both the transmembrane domain A-site and the B-site in both CDFs results in a cellular metal sensitivity similar to that of the corresponding null-mutants. However, multiple mutations in the predicted cytoplasmic C1/C2 cluster of MntE have no impact on cellular Mn resistance, in contrast to the analogous substitutions in CzcD for Zn resistance. Deletion of the MntE-specific C-terminal tail, present only in Mn-specific bacterial CDFs, resulted in only a modest growth phenotype. Further analysis of MntE-CzcD functional chimeric transporters showed that Asn and Asp in the N: D- D: D A-site motif of MntE and the most N-terminal His in H: D-HD site A of CzcD, play key roles in transporter metal selectivity.


Cation diffusion facilitator (CDF) proteins are divalent metal ion transporters conserved from bacteria to humans that play important roles in cellular physiology, from metal homeostasis and resistance to type I diabetes in vertebrates. The respiratory pathogen Streptococcus pneumoniae expresses two metal CDF transporters CzcD and MntE. How CDFs achieve metal selectivity is unclear. We show here that CzcD and MntE are true paralogs, as CzcD transports zinc, while MntE selectively transports manganese. Through the use of an extensive collection of pneumococcal variants, we show that a primary determinant for metal selectivity is the A-site within the transmembrane domain. This extends our understanding of how CDFs discriminate among transition metals.

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PMID: 26787764 [PubMed - as supplied by publisher]