Identification of the primary metal ion-activation sites of the diphtheria tox represser by X-ray crystallography and site-directed mutational analysis

Abstract

The diphtheria tox repressor, DtxR, is a 226 amino acid transition metal ion-activated regulatory protein that controls the expression of diphtheria toxin in toxigenic Corynebacterium diphtheriae. The previously solved three-dimensional DtxR structures have identified two potential metal ion binding sites which may play a role in the activation of DNA binding by the repressor. We have used both X-ray crystallographic and site-directed mutational analysis of DtxR(C102D)-Ni2+ complexes and DtxR to identify the metal ion-binding site which results in the activation of the repressor. We demonstrate that DtxR contains both a primary and an ancillary metal ion binding site. The primary site functions directly in the activation of DNA binding. In contrast, the ancillary site contributes weakly, if at all, to activation.

Metal-Linked Dimerization in the Iron-Dependent Regulator from Mycobacterium tuberculosis

“…Initial crystal structures clearly indicated two metal binding sites per monomer in either protein (7,9,10,(24)(25)(26). The functional significance of these sites for repressor function have been thoroughly investigated in DtxR (9,(27)(28)(29). More recently, a third metal binding site located in the SH3 domain was identified in the crystal structure of IdeR (6,7), although the biological relevance of this site has not been established.…”

“…The energetics and stoichiometry of metal binding by DtxR and IdeR have been a topic of interest for more than a decade. Initial crystal structures clearly indicated two metal binding sites per monomer in either protein (7,9,10,(24)(25)(26). The functional significance of these sites for repressor function have been thoroughly investigated in DtxR (9,(27)(28)(29).…”

“…One metal binding site is formed entirely by the residues from the N-terminal domain (M10, C102, E105, and H106) ( Figure 1). Mutating any one of these residues to alanine abrogates repressor activity in DtxR (9); this binding site is referred to as the primary metal binding site. A second metal binding site is formed by residues from the N-terminal domain and by two residues from the C-terminal SH3-like domain (H79, E83, H98, E172, and Q175).…”

“…A second metal binding site is formed by residues from the N-terminal domain and by two residues from the C-terminal SH3-like domain (H79, E83, H98, E172, and Q175). Mutating any one of these residues in DtxR reduces but does not abolish repressor activity (9); consequently, this site is referred to as the ancillary site. The primary and ancillary metal binding sites are separated by ∼9 Å and interact via a series of hydrogen bonds and salt bridges in the metal bound form (10), which suggests that a functional cooperativity may exist between these two sites.…”

Comparison of high‐resolution structures of the diphtheria toxin repressor in complex with cobalt and zinc at the cation‐anion binding site

“…The second transition metal-binding site in wild-type DtxR has so far only been observed in the Cd-DtxR (Qiu et al, 1995) and Mn-DtxR structures (Qiu et al, 1996). In the crystal structure of the Cysl02Asp DtxR variant, determined by Ding et al (1996), a nickel-binding site is described near site 2 in the wild-type structure. The present paper focuses on the comparison of the zinc-and cobalt-containing repressors.…”