This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Song, L.
Right arrow Articles by Summers, A. O.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Song, L.
Right arrow Articles by Summers, A. O.

 Previous Article  |  Next Article 

Journal of Bacteriology, March 2004, p. 1861-1868, Vol. 186, No. 6
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.6.1861-1868.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Engineered Single-Chain, Antiparallel, Coiled Coil Mimics the MerR Metal Binding Site

Lingyun Song,1,2 Jonathan Caguiat,1,2,{dagger} Zhongrui Li,2,3 Jacob Shokes,2,3 Robert A. Scott,2,3 Lynda Olliff,1,2 and Anne O. Summers1,2*

Department of Microbiology,1 Department of Chemistry,3 Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 306022

Received 14 August 2003/ Accepted 21 November 2003

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 203Hg(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.

* Corresponding author. Mailing address: Department of Microbiology, The University of Georgia, Athens, GA 30602-2605. Phone: (706) 542-2669. Fax: (706) 542-6140. E-mail: summers{at}uga.edu.

{dagger} Present address: NT, Inc., Detroit, MI 48202.

Journal of Bacteriology, March 2004, p. 1861-1868, Vol. 186, No. 6
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.6.1861-1868.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Qin, J., Song, L., Brim, H., Daly, M. J., Summers, A. O. (2006). Hg(II) sequestration and protection by the MerR metal-binding domain (MBD).. Microbiology 152: 709-719 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»