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. 2008 May 30;283(22):15142-51.
doi: 10.1074/jbc.M800591200. Epub 2008 Mar 25.

A Histidine-Rich and Cysteine-Rich Metal-Binding Domain at the C Terminus of Heat Shock Protein A From Helicobacter Pylori: Implication for Nickel Homeostasis and Bismuth Susceptibility

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A Histidine-Rich and Cysteine-Rich Metal-Binding Domain at the C Terminus of Heat Shock Protein A From Helicobacter Pylori: Implication for Nickel Homeostasis and Bismuth Susceptibility

Shujian Cun et al. J Biol Chem. .
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Abstract

HspA, a member of the GroES chaperonin family, is a small protein found in Helicobacter pylori with a unique histidine- and cysteine-rich domain at the C terminus. In this work, we overexpressed, purified, and characterized this protein both in vitro and in vivo. The apo form of the protein binds 2.10 +/- 0.07 Ni(2+) or 1.98 +/- 0.08 Bi(3+) ions/monomer with a dissociation constant (K(d)) of 1.1 or 5.9 x 10(-19) microm, respectively. Importantly, Ni(2+) can reversibly bind to the protein, as the bound nickel can be released either in the presence of a chelating ligand, e.g. EDTA, or at an acidic pH (pH((1/2)) 3.8 +/- 0.2). In contrast, Bi(3+) binds almost irreversibly to the protein. Both gel filtration chromatography and native electrophoresis demonstrated that apo-HspA exists as a heptamer in solution. Unexpectedly, binding of Bi(3+) to the protein altered its quaternary structure from a heptamer to a dimer, indicating that bismuth may interfere with the biological functions of HspA. When cultured in Ni(2+)-supplemented M9 minimal medium, Escherichia coli BL21(DE3) cells expressing wild-type HspA or the C-terminal deletion mutant clearly indicated that the C terminus might protect cells from high concentrations of external Ni(2+). However, an opposite phenomenon was observed when the same E. coli hosts were grown in Bi(3+)-supplemented medium. HspA may therefore play a dual role: to facilitate nickel acquisition by donating Ni(2+) to appropriate proteins in a nickel-deficient environment and to carry out detoxification via sequestration of excess nickel. Meanwhile, HspA can be a potential target of the bismuth antiulcer drug against H. pylori.

Figures

FIGURE 1.
FIGURE 1.
Sequence alignment of H. pylori HspA with homologues from other pathogenic microorganisms (A) or model species (B). Identical residues are marked with a black background, and conserved ones are shaded in light gray. The sequence alignment was done by ClustalW (version 1.83), and the sequence similarities were calculated by the Needle program from the EMBOSS package (version 3.0) using the H. pylori homologue as the template. The following sequences were retrieved from the GenBank™ Protein Databank. A, HspA (H. pylori; AAP51175.1), co-chaperonin GroES (Helicobacter hepaticus; NP_860731), co-chaperonin GroES (Leifsonia xyli xyli; YP_062808), 10-kDa chaperonin (Propionibacterium acnes; YP_056460), co-chaperonin GroES (Streptomyces coelicolor; NP_628919), GroES (Wigglesworthia glossinidia; AAK07426), chaperonin GroS (Mycobacterium smegmatis; YP_885961), co-chaperonin GroES (Yersinia pestis; NP_403998), co-chaperonin GroES (E. coli; NP_418566.1), co-chaperonin GroES (Photorhabdus luminescen; NP_931325), co-chaperonin GroES (Salmonella enterica; YP_219195), co-chaperonin GroES (Haemophilus influenzae; NP_438700), and co-chaperonin GroES (Pasteurella multocida; NP_246043). B, heat shock 10-kDa protein (Sus scrofa; AAP32465), heat shock 10-kDa protein (Homo sapiens; NP_002148), chaperonin 10 (Rattus norvegicus; AAC53361.1), chaperonin 10 (Mus musculus; AAF67345.1), chaperonin 10 (Gallus gallus; NP_990398.1), chaperonin 10 (Xenopus tropicalis; AAH77653.1), chaperonin 10 (Danio rerio; AAH71419.1), GA10877-PA (Drosophila pseudoobscura; EAL31011.1), and HspA (H. pylori; AAP51175.1).
FIGURE 2.
FIGURE 2.
UV-visible difference spectra of apo-HspA (38 μm in 20 mm Hepes buffer, pH 7. 4, in the presence of 6-fold tris(2-carboxyethyl)phosphine hydrochloride at room temperature) after the addition of different molar equivalents of NiSO4 (A) or Bi-NTA (B). The insets are titration curves plotted at 316 nm for Ni2+ binding and 364 nm for Bi3+ binding, indicative of binding two metal ions/monomer.
FIGURE 3.
FIGURE 3.
Binding profiles of HspA by equilibrium dialysis. HspA (20 μm in 300 μl of protein solution) was dialyzed against 100 ml of buffer containing 50 mm Tris-HCl and 100 mm NaCl at pH 7.2 with serial concentrations of NiSO4 (A) or Bi-NTA (B) at room temperature overnight. The graphs show Hill plots of the molar ratios of bound Ni2+/Bi3+ to HspA against concentrations of metals in the dialysis buffer.
FIGURE 4.
FIGURE 4.
pH-dependent profiles of Ni2+/Bi3+ release from metal-bound HspA. Metal-saturated HspA in 20 mm Hepes buffer was used for pH titration and monitored by the decrease in absorbance of the peaks at 316 nm for Ni2+-HspA (▪) and 364 nm for Bi3+-HspA (▴).
FIGURE 5.
FIGURE 5.
Kinetics of metal release from HspA. Time-dependent absorbance at 316 nm for Ni2+-HspA (▪) or 364 nm for Bi3+-HspA (▴) was recorded for Ni2+/Bi3+-saturated HspA upon the addition of 40 molar eq of EDTA, pH 7.4, at room temperature. The absorbance at each time point was normalized to percentages to represent the amount of bound metals.
FIGURE 6.
FIGURE 6.
Analysis of the oligomeric states of apo- and metal-bound HspA by gel filtration chromatography and native electrophoresis. A, gel filtration profiles for apo-, nickel-, and bismuth-bound HspA on a Superdex 75 10/300 GL column eluted with a buffer containing 50 mm Tris-HCl and 100 mm NaCl, pH 7.0, at room temperature. Note that bismuth induced protein quaternary structure changes from a heptamer to a dimer. mAU, milli-absorbance units. B, native electrophoresis for apo-, nickel-, and bismuth-bound HspA. The electrophoresis was carried out in 50 mm Tris and 200 mm glycine, pH 8.8, at room temperature.
FIGURE 7.
FIGURE 7.
Effects of nickel and bismuth on the growth of E. coli cells expressing wild-type HspA (closed bars) or the C-terminal deletion HspA (open bars). Metal susceptibility is presented by bacterial growth in M9 minimal medium supplemented with NiSO4 (A) or CBS (B). The A550 of E. coli culture was measured as a growth indicator. The growth rate between the wild type and mutant was statistically compared by Student's t test, and asterisks indicate that the two sets of data are significantly different from each other. Each datum point was determined for independently cultured bacteria in triplicate, presented as the mean ± S.D.
FIGURE 8.
FIGURE 8.
Growth curves of E. coli cells expressing wild-type HspA or C-terminal deletion mutant. E. coli cells were grown in M9 minimal medium supplemented with 5 μm NiSO4 (A) or 500 μm CBS (B). At each selected time point, the ratios of the A550 values at different times versus the value at starter culture (t = 0 h) were used to represent the relative growth rate. The statistical difference between the wild type (WT) and mutant (MT) was estimated by Student's t test, and asterisks indicated that the two sets of data are significantly different from each other. Each datum point was tested independently in triplicate, presented as the mean ± S.D.
FIGURE 9.
FIGURE 9.
Proposed scheme presenting the oligomeric state of HspA in the presence of nickel or bismuth. The front subunit is highlighted, and the rest are shaded in gray. The protein structure of H. pylori HspA was modeled based on the crystal structure of Mycobacterium tuberculosis GroES (Protein Data Bank entry 1hx5), except for the C-terminal domain due to lack of a modeling template. Ni2+ and Bi3+ presumably coordinate to different residual ligands. It is also worth mentioning that the C-terminal deletion mutant is unable to carry detectable Ni2+ (S. Cun, H. Li, and H. Sun, unpublished data), and it may be immune to the structural disruption induced by Bi3+ binding if the given speculation is real.

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