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Infection and Immunity, April 1999, p. 1806-1811, Vol. 67, No. 4
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Anion-Coordinating Residues at Binding Site 1 Are Essential for the Biological Activity of the Diphtheria Toxin Repressor

Joanne Goranson-Siekierke,1 Ehmke Pohl,2 Wim G. J. Hol,2,3 and Randall K. Holmes1,*

Department of Microbiology, University of Colorado Health Sciences Center, Denver, Colorado 80262,1 and Departments of Biological Structure and Biochemistry, Biomolecular Structure Program,2 and Howard Hughes Medical Institute,3 University of Washington, Seattle, Washington 98195

Received 14 October 1998/Returned for modification 17 December 1998/Accepted 31 December 1998

The homodimeric diphtheria toxin repressor (DtxR) uses Fe2+ as a corepressor, binds to iron-regulated promoters, and negatively regulates the syntheses of diphtheria toxin, corynebacterial siderophore, and several other Corynebacterium diphtheriae products. The crystal structure of DtxR shows that the second domain of each monomer has two binding sites for Fe2+ or certain other divalent metal ions. In addition, site 1 binds a sulfate or phosphate anion, suggesting that phosphate may function intracellularly as a co-corepressor. The effects of alanine substitutions for selected residues in sites 1 and 2 were determined by measuring the beta -galactosidase activities of a tox operator/promoter-lacZ reporter construct in Escherichia coli strains expressing each DtxR variant. Our studies demonstrated that single alanine substitutions for the anion-binding residues in site 1 (R80A, S126A, or N130A) caused severely decreased DtxR activity, similar to the effects of alanine substitutions for metal-binding residues in site 2 (C102A, E105A, or H106A) and greater than the effects of alanine substitutions for metal-binding residues in site 1 (H79A, E83A, or H98A) reported previously by other investigators. Various combinations of alanine substitutions for site 1 and site 2 residues were also analyzed to further elucidate the roles of these cation- and anion-binding ligands in DtxR activity. Furthermore, the interaction between residue E20 in the DNA binding domain and R80 in anion/cation binding site 1 was analyzed, and the E20A variant of DtxR was shown to have a phenotype indistinguishable from that of the R80A variant. Our data demonstrated for the first time that the anion-binding residues R80, S126, and N130 at site 1 are essential for DtxR activity. The data also showed that the interaction of E20 in domain 1 with R80 in domain 2, first revealed by X-ray crystallography in apo-DtxR and holo-DtxR, is a structural feature of DtxR that is important for its repressor activity.

* Corresponding author. Mailing address: Department of Microbiology, B-175, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver, CO 80262. Phone: (303) 315-7903. Fax: (303) 315-6785. E-mail: Randall.Holmes{at}UCHSC.edu.

Infection and Immunity, April 1999, p. 1806-1811, Vol. 67, No. 4
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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Copyright © 1999 by the American Society for Microbiology. All rights reserved.