Engineered single-chain, antiparallel, coiled coil mimics the MerR metal binding site

Abstract

The repressor-activator MerR that controls transcription of the mercury resistance (mer) operon is unusual for its high sensitivity and specificity for Hg(II) in in vivo and in vitro transcriptional assays. The metal-recognition domain of MerR resides at the homodimer interface in a novel antiparallel arrangement of alpha-helix 5 that forms a coiled-coil motif. To facilitate the study of this novel metal binding motif, we assembled this antiparallel coiled coil into a single chain by directly fusing two copies of the 48-residue alpha-helix 5 of MerR. The resulting 107-residue polypeptide, called the metal binding domain (MBD), and wild-type MerR were overproduced and purified, and their metal-binding properties were determined in vivo and in vitro. In vitro MBD bound ca. 1.0 equivalent of Hg(II) per pair of binding sites, just as MerR does, and it showed only a slightly lower affinity for Hg(II) than did MerR. Extended X-ray absorption fine structure data showed that MBD has essentially the same Hg(II) coordination environment as MerR. In vivo, cells overexpressing MBD accumulated 70 to 100% more (203)Hg(II) than cells bearing the vector alone, without deleterious effects on cell growth. Both MerR and MBD variously bound other thiophilic metal ions, including Cd(II), Zn(II), Pb(II), and As(III), in vitro and in vivo. We conclude that (i) it is possible to simulate in a single polypeptide chain the in vitro and in vivo metal-binding ability of dimeric, full-length MerR and (ii) MerR's specificity in transcriptional activation does not reside solely in the metal-binding step.

FIG. 1.

Construction of MBD. The MBD gene was constructed by connecting two α-helices 5 of MerR in tandem with three nonnative amino acids, SSG, as a bridge. The carboxyl terminus of MBD was fused with the 10-amino-acid Strep affinity tag, represented in purple, in the pASK-IBA3 vector. A Strep-tag fusion of full-length MerR was also made with this vector. Bars indicate helices as follows: blue bars indicate α-helices in the metal binding domain of MerR, and gray bars indicate other α-helices of MerR. Lines indicate non-α-helix regions as follows: the red line indicates the SSG linker, and the blue and green lines indicate the loop after α-helix 5 and the region after the loop, respectively. Orange dots indicate cysteines involved in Hg(II) binding, which are also given in orange in the sequences.

FIG. 2.

Equilibrium ultrafiltration determination of ²⁰³Hg binding to purified MerR and MBD. The values are the means of duplicate measurements. The average standard deviation was 10%. MerR values are presented as per mole of MerR dimers.

FIG. 3.

Accumulation of ²⁰³Hg(II) by cells containing MerR or MBD. The values are the means of duplicate measurements. The average standard deviation was 29%. The units [moles of ²⁰³Hg(II) per picomole per unit of cell mass] indicate the amount of ²⁰³Hg(II) in cell cultures normalized on the basis of OD₆₀₀.

FIG. 4.

EXAFS analysis of Hg(II) complexes with MerR and MBD. XAS edge (top) and EXAFS FT and experimental k3-weighted EXAFS spectra (bottom and insert, respectively) for comparison of full-length MerR-Strep-tag (solid lines) and MBD-Strep-tag (dashed lines) are shown. The conditions used are shown in Table 1, and curve-fitting results are shown in Table 2.

FIG. 5.

Binding of other thiophilic metals by MBD and MerR. (A) In vitro. (B) In vivo. The values are the means of duplicate measurements. Average standard deviations were 28% for panel A and 9% for panel B.