DtxR dimer When Corynebacterium diphtheriae encounters an environment with a low concentration of iron ions, it initiates the synthesis of siderophores, as well as diphtheria toxin. The diphtheria toxin repressor (DtxR) plays a key role in this Fe(II)-dependent, global regulatory system and is the prototype for a new family of Fe-dependent repressor proteins in gram positive bacteria.
The crystal structure of dimeric DtxR holo-repressor in complex with different transition metals shows that each subunit consists of an N-terminal DNA-binding domain, followed by an interface domain that contains the metal binding sites, and a third, very flexible C-terminal domain. Each DNA-binding domain contains a typical helix-turn-helix motif and has a topology which is very similar to catabolite gene activator protein (CAP). Molecular modeling suggests that bound DNA adopts a bent conformation with helices 3 of DtxR interacting with the major grooves.
DtxR dimer The crystal structure of the DtxR holo-repressor suggests that the divalent cation co-repressor controls motions of the DNA binding domain. In this way the metal co-repressor governs the distance between operator recognition elements in the two subunits and, consequently, DNA recognition.
The major hurdle in the structure determination was the introduction of heavy atom derivatives to the crystals. We have tried soaking the crystals with over 50 heavy atom compounds, as well as co-crystallization of the repressor in the pressence of CdCl2 or HgCl2. After 100 data sets were collected in house or at synchrotron sources, a few derivatives were identified. Because they are relatively weak and sharing a couple of common sites, MIRAS phasing with nine derivatives was necessary for obtaining the final interpretable electron density maps. The overall figure of merit was 0.60 for the reflections from 50.0 to 2.8Å. Density modification including solvent flattening, histogram matching and applying Sayre's equation were carried out, and the resulting electron density map was clearly traced, mostly including side chains. The structures of Co-DtxR and Mn-DtxR are now refined to a R-factors of 17% at 2.0Å, 16% & at 2.2Å resolution respectively. The structure of the flexible third domain could be determined at this high resolution. This domain exhibits a fold very similar to the SH3 domain. The rms deviation of 46 equivalent CA atoms is 3 Å, and the sequence identity is only 7 %.
Crystals in the presence of Fe++, Co++, Ni++, Zn++, Mn++, Cd++ ( all co-repressors of DtxR in vitro ) were isomorphous to the native crystal and were determined. There are no significant conformational changes between the crystal structures of DtxR with the different metals and the crystal structure of Apo-DtxR. The structure in a different crystal form with a doubled c-axis (c = 216Å) was solved by molecular replacement. In this crystal form the crystallographic two-fold axis has slightly shifted converting it into a non-crystallographic two-fold. Thus, the spacegroup is P3221. The structures are virtually identical in the two closely related crystal forms. Recently, we were able to overexpress and crystallize the Cys102Ser DtxR variant. This mutant has a reduced activity in DNA-binding gel-shift assays. The crystal structure is very similar to the wildtype structure. No significant conformational changes were observed. Metal binding site I is occupied with a metal, whereas metal binding site II is empty. This indicates an important role of site II for metal-activated DNA-binding.