Zinc is considered to be an essential micronutrient despite the fact that it is present in bacterial cells at close to millimolar concentrations. However, whilst the total concentration of this redox-insensitive ion is quite high, essentially none of the million or so atoms in a typical Escherichia coli
cell exist free in solution (Outten & O’Halloran, 2001
). This vanishingly low concentration of free zinc represents an interesting challenge for the assorted intracellular and membranal systems concerned with zinc homeostasis. These include proteins and other biomolecules that participate in the sequestration and the import and export of zinc to ensure that it properly serves its vital catalytic, regulatory and structural roles (Hantke, 2005
; Blencowe & Morby, 2003
; Choudhury & Srivastava, 2001
One group of proteins that are involved in zinc export belong to the cation-diffusion facilitator (CDF) family (Paulsen & Saier, 1997
). The CDFs are ubiquitous in nature, with representatives in the three taxonomic domains archaea, bacteria and eukaryota. Some examples include FieF in E. coli
(Grass et al.
), CzcD in Alcaligenes eutrophus
CH34 (since renamed Wautersia metallidurans
CH34; Nies et al.
; Munkelt et al.
), Zrc1 in Saccharomyces cerevisiae
(Conklin et al.
), AtZat in Arabidopsis thaliana
(Bloss et al.
) and ZnTX in mammals (Haney et al.
). In Gram-negative bacteria, CDF proteins move zinc and other metals such as cobalt and manganese from the cytoplasm into the periplasmic space by means of a proton/cation antiporter mechanism (Haney et al.
). Despite the fact that no three-dimensional structure of a representative from this family is yet available, sequence alignment and biochemical and computational analyses reveal common features. These include six membrane-spanning α-helices per monomer, histidine-rich clusters in the N- and C-termini and a tendency to dimerize (Paulsen & Saier, 1997
; Haney et al.
; Spada et al.
CzrB (cadmium-zinc resistance protein B) was the first CDF family member to be identified in Thermus thermophilus
(Spada et al.
; Kolaj et al.
). As with other CDF transporters, this 291-residue protein has a putative transmembrane hexahelical domain. However, it lacks the hallmark histidine-rich N- and C-termini. Interestingly, it has a 92-amino-acid C-terminus that may form an independent extramembranal cytosolic domain. In support of this, the czrB
gene has been shown to contain an ATG consensus sequence at Met200, with a putative ribosome-binding site nearby. This C-terminal domain is proposed to play a role in metal-ion sequestration and transport as a soluble cytoplasmic fragment (sf-CzrB; Spada et al.
). The czrB
gene conferred a slightly enhanced tolerance for zinc on E. coli
. Surprisingly, both the czrB
genes were found to support the growth of E. coli
cultures to higher densities during bacteriophage and recombinant protein production (Spada et al.
). This suggested an ill-defined chaperone-like function for the corresponding gene products.
Here, we describe the production, purification, crystallization and preliminary X-ray analysis of the soluble C-terminal fragment of CzrB in its apo and zinc-loaded forms.