The diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae regu
lates the expression of the gene on corynebacteriophages that encodes dipht
heria toxin (DT). Other genes regulated by DtxR include those that encode p
roteins involved in siderophore-mediated iron uptake. DtxR requires activat
ion by divalent metals and holo-DtxR is a dimeric regulator with two distin
ct metal-binding sites per three-domain monomer. At site 1, three side chai
ns and a sulfate or phosphate anion are involved in metal coordination. In
the DtxR-DNA complex this anion is replaced by the side chain of Glu170 pro
vided by the third domain of the repressor. At site 2 the metal ion is coor
dinated exclusively by constituents of the polypeptide chain. In this paper
, five crystal structures of three DtxR variants focusing on residues Glu20
, Arg80 and Cys102 are reported. The resolution of these structures ranges
from 2.3 to 2.8 Angstrom. The side chain of Glu20 provided by the DNA-bindi
ng domain forms a salt bridge to Arg80, which in turn interacts with the an
ion. Replacing either of the salt-bridge partners with an alanine reduces r
epressor activity substantially and it has been inferred that the salt brid
ge could possibly control the wedge angle between the DNA-binding domain an
d the dimerization domain, thereby modulating repressor activity. Cys102 is
a key residue of metal site 2 and its substitution into a serine abolishes
repressor activity. The crystal structures of Zn-Glu20Ala-DtxR, Zn-Arg80Al
a-DtxR, Cd-Cys102Ser-DtxR and apo-Cys102Ser-DtxR in two related space group
s reveal that none of these substitutions leads to dramatic rearrangements
of the DtxR fold. However, the five crystal structures presented here show
significant local changes and a considerable degree of flexibility of the D
NA-binding domain with respect to the dimerization domain. Furthermore, all
five structures deviate significantly from the structure in the DtxR-DNA c
omplex with respect to overall domain orientation. These results confirm th
e importance of the hinge motion for repressor activity. Since the third do
main has often been invisible in previous crystal structures of DtxR, it is
also noteworthy that the SH3-like domain could be traced in four of the fi
ve crystal structures.