Metal stoichiometry and functional studies of the diphtheria toxin repressor

doi:10.1073/pnas.0737977100

Metal stoichiometry and functional studies of the diphtheria toxin repressor

^*Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109; ^†Departments of Biochemistry and Chemistry and ^‡Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454; ^§Department of Medicine, Boston University School of Medicine, Boston, MA 02118; and ^¶Departments of Chemistry and Molecular and Cell Biology, University of California, Berkeley, CA 94720

Edited by Jack Halpern, University of Chicago, Chicago, IL, and approved February 12, 2003 (received for review December 30, 2002)

Abstract

Diphtheria toxin repressor (DtxR) is a transition metal ion-activated repressor in Corynebacterium diphtheriae. DtxR is an iron sensor; metal-bound DtxR represses transcription of genes downstream of the tox operator. Wild-type DtxR [DtxR(wt)] and several mutant forms were overexpressed and purified from Escherichia coli. DtxR was isolated without bound metal. Metal reconstitution gave a binding stoichiometry of 2 per monomer for DtxR(wt) and 1 per monomer for DtxR(H79A) and DtxR(M10A). DNA binding of DtxR(H79A) and DtxR(M10A) indicates that metal site 2 is essential for activity. Metal binding lowers the dimerization K _d of DtxR from low micromolar to 33 nM. Gel electrophoretic mobility-shift assays show that Fe²⁺ and not Fe³⁺ activates DtxR for DNA binding. This finding suggests that gene regulation by DtxR may be sensitive not only to iron levels but also to redox state of the iron. Mutations in the tox operator sequence indicate that DtxR dimers binding to DNA may be highly cooperative.

Footnotes

↵ ‖ To whom correspondence should be addressed at: Department of Chemistry, University of California, Berkeley, CA 94720. E-mail: marletta{at}cchem.berkeley.edu.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

DtxR,

diphtheria toxin repressor;

DtxR(wt),

wild-type DtxR;

ferrozine,

3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine;

QAA,

quantitative amino acid analysis;

EMSA,

gel electrophoretic mobility-shift assay;

ICP–MS,

inductively coupled plasma high-resolution mass spectrometer;

SPR,

surface plasmon resonance