Overexpression, purification, and site-directed spin labeling of the Nramp metal transporter from Mycobacterium leprae

Abstract

It has long been recognized that the pathogenicity of a broad range of intracellular parasites depends on the availability of transition metal ions, especially iron. Nramp1 (natural resistance-associated macrophage protein 1), a proton-coupled divalent metal ion transporter, has been identified as a controlling factor in the resistance or susceptibility to infection with a diverse range of intracellular pathogens such as Toxoplasma, Salmonella, Mycobacterium, and Leishmania. The role of divalent metal ion transport is even more compelling given the existence of Nramp homologs in several intracellular parasites, such as mycobacteria. We have confirmed the functional homology of the Nramp homologue from Mycobacterium leprae by using a yeast complementation assay for divalent cation uptake. To facilitate a concerted biochemical and structural analysis of this important class of transporters, the M. leprae Nramp was expressed in Escherichia coli. Dual affinity tags were engineered at the N and C termini to allow for isolation of full-length protein at >95% purity. Site-directed spin labeling of Cys-299 reveals a flexible hinge-like domain. A weak dipolar interaction is detected between the nitroxide and paramagnetic transition ions, indicating this position is approximately 19 A from the nearest high affinity binding site.

Figure 1

Functional complementation of the yeast SMF null mutant (ΔSMF3) by the Ml MntH gene. The various yeast strains were grown on YPD-agar plates as described in Materials and Methods. The medium in the right plate was supplemented by 3 mM EGTA. (A) Wild-type strain W 303. (B) ΔSMF3 strain. (C) ΔSMF3 strain transformed with the yeast expression vector pBFG1. (D) ΔSMF3 strain transformed with pBFG1 containing the cloned Ml MntH.

Figure 2

A topology model for the Ml MntH protein. The approximate sequence positions for residues at the bilayer interface are numerically indicated. The relative positions of the conserved charged residues (+ or −) and histidine residues (H) are also indicated. The location of Strep II (ST) and E tag (ET) affinity sequences in the fusion protein are noted; however, the residue numbering is given according to the native sequence. The prediction places three acidic and three basic residues within membrane spanning regions, suggesting the presence of ion pairs within the bilayer. The approximate position of Gly-95, the conserved glycine residue of TM IV found mutated in Nramp1 and Nramp2-responsible defects, is indicated.

Figure 3

(A) Inducible expression of Ml MntH in the membrane fraction of E. coli. Shown is the Western blot analysis of expression from the gene cloned into the pBAD-TOPO plasmid containing the C-terminal V5 peptide epitope. The positions of protein molecular mass markers are indicated to the left in kDa. Lane 1, membranes from uninduced cells; lane 2, membranes from cells induced with l-arabinose. (B) SDS/PAGE gel of purified Ml MntH protein after affinity chromatography with the StrepTag and then E tag columns. The purified sample was loaded into lane 1. Lane 2 shows the molecular size standards in kDa. (C) Ml MntH-dependent ⁵⁴Mn uptake. Uptake of radioactivity in uninduced (U) and induced (I) cells after 10 min of incubation. Levels of accumulation were determined from four individual samples measured for each time point. Each value represents the mean ± SE of three experiments performed in separate cultures.

Figure 4

Slot blot expression analysis of the Ml MntH protein as a function of metal ion presence in the growth media during induction. (A) Detergent-solubilized membranes from cells transformed with pBAD-mycHis plasmid (control). (B) Detergent-solubilized membranes from cells transformed with pBAD-mycHis plasmid containing the Ml MntH gene. (C) Same as B, but with 1 mM Mn²⁺, Zn²⁺, and Fe²⁺ in the growth media. Detection was performed by using the anti-E tag antibody. Each slot was loaded with 300 μg total membrane protein.

Figure 5

The EPR line shape of position 299 in the Ml MntH protein in DDM micelles (A) and after reconstitution into a lipid bilayer (B). In A, the distance of position 299 to the nearest metal binding site was measured by adding 0.5 mM CuCl₂ (solid dark line), which will broaden the spectrum in a distance-dependent manner. The control spectrum (broken gray line) was obtained in the presence of 0.5 mM ZnCl₂.