3glx

Crystal Structure Analysis of the DtxR(E175K) complexed with Ni(II)

spinqualitylabelsall models 3glx, resolution 1.85Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 3glx is a 1 chain structure with sequence from Corynebacterium diphtheriae. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 1.85Å Ligands: NI, PO4 Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function DTXR_CORDI Iron-binding repressor of the dipheteria toxin gene expression. May serve as a global regulator of gene expression. Represses ripA under iron excess. Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMed The metal-ion-activated diphtheria toxin repressor (DtxR) is responsible for the regulation of virulence and other genes in Corynebacterium diphtheriae. A single point mutation in DtxR, DtxR(E175K), causes this mutant repressor to have a hyperactive phenotype. Mice infected with Mycobacterium tuberculosis transformed with plasmids carrying this mutant gene show reduced signs of the tuberculosis infection. Corynebacterial DtxR is able to complement mycobacterial IdeR and vice versa. To date, an explanation for the hyperactivity of DtxR(E175K) has remained elusive. In an attempt to address this issue, we have solved the first crystal structure of DtxR(E175K) and characterized this mutant using circular dichroism, isothermal titration calorimetry, and other biochemical techniques. The results show that although DtxR(E175K) and the wild type have similar secondary structures, DtxR(E175K) gains additional thermostability upon activation with metal ions, which may lead to this mutant requiring a lower concentration of metal ions to reach the same levels of thermostability as the wild-type protein. The E175K mutation causes binding site 1 to retain metal ion bound at all times, which can only be removed by incubation with an ion chelator. The crystal structure of DtxR(E175K) shows an empty binding site 2 without evidence of oxidation of Cys102. The association constant for this low-affinity binding site of DtxR(E175K) obtained from calorimetric titration with Ni(II) is K(a)=7.6+/-0.5x10(4), which is very similar to the reported value for the wild-type repressor, K(a)=6.3x10(4). Both the wild type and DtxR(E175K) require the same amount of metal ion to produce a shift in the electrophoretic mobility shift assay, but unlike the wild type, DtxR(E175K) binding to its cognate DNA [tox promoter-operator (toxPO)] does not require metal-ion supplementation in the running buffer. In the timescale of these experiments, the Mn(II)-DtxR(E175K)-toxPO complex is insensitive to changes in the environmental cation concentrations. In addition to Mn(II), Ni(II), Co(II), Cd(II), and Zn(II) are able to sustain the hyperactive phenotype. These results demonstrate a prominent role of binding site 1 in the activation of DtxR and support the hypothesis that DtxR(E175K) attenuates the expression of virulence due to the decreased ability of the Me(II)-DtxR(E175K)-toxPO complex to dissociate at low concentrations of metal ions. Decreased sensitivity to changes in the concentration of metal ions as the basis for the hyperactivity of DtxR(E175K).,D'Aquino JA, Denninger AR, Moulin AG, D'Aquino KE, Ringe D J Mol Biol. 2009 Jul 3;390(1):112-23. Epub 2009 May 8. PMID:19433095 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also Diphtheria toxin repressor

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