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

Pohl, Ehmke; Qiu, Xiayang; Must, Lisa M.; Holmes, Randall K.; Hól, Wim G. J.

doi:10.1002/pro.5560060519

Cited by 39 publications

(55 citation statements)

References 24 publications

(28 reference statements)

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“…Metal Site 1-As mentioned before, metal binding site 1 has been observed to be occupied in all high resolution crystal structures of DtxR determined in the presence of divalent metal ions so far (21,23,24 (Fig. 1A).…”

Section: Table IV Pairwise Comparison Of the Apo-and Zn-dtxr Structuressupporting

confidence: 61%

“…In evaluating these results, it may be useful to note that the modification of Cys 102 , which is observed time and again in high resolution metal-containing wild type DtxR structures (23,24), may be preventing us from unraveling the structure of the true holo-repressor, which should contain two fully occupied metal binding sites and not one as has been the case in virtually all wild type DtxR structures reported so far. The possibility cannot be excluded that in the true holo-repressor, with an unmodified Cys 102 , the DNA binding domains are even more differently positioned with respect to the DtxR core domains than has been observed in the apo-and metal-containing DtxR structures reported in this paper.…”

Section: Discussionmentioning

confidence: 99%

“…All other data sets were collected at cryogenic temperatures using crystals frozen in rayon loops (26). Before cryo-cooling, the crystals were transferred into a drop containing 1.2-1.5 M ammonium sulfate and 15-20% glycerol (24). The data sets for apo-DtxR and for crystal form I of Zn-DtxR were collected on an RAXIS-II imaging plate detector using monochromatic CuK␣ radiation and focusing mirrors.…”

Section: Methodsmentioning

confidence: 99%

“…It has been suggested that the lack of metal binding at site 2 is due to the formation of a persulfide or a mixed disulfide at cysteine 102 (23,24). In all metal-bound DtxR crystal structures investigated so far, including the two Zn-DtxR structures described in this paper, the F o Ϫ F c difference electron density had its strongest peak at approximately 2 Å from the S ␥ of Cys 102 , which, based on geometric criteria, can be interpreted as a sulfur atom bound to the S ␥ (23,24). However, in the present study, this peak is significantly weaker in apo-DtxR crystal form I and not present in crystal form II of apo-DtxR.…”

Section: Table IV Pairwise Comparison Of the Apo-and Zn-dtxr Structuresmentioning

confidence: 99%

“…Crystal structures of wild-type DtxR in complex with different divalent transition metals have been determined at 2.8-Å resolution (21) and of apo-DtxR at 3.0-Å resolution in a different space group (22). Recently, the resolution for Co-DtxR, Mn-DtxR, and Zn-DtxR has been extended to 1.85, 2.2, and 2.4 Å, respectively (23,24). These structures reveal an N-terminal domain of residues 1-73 that includes the DNA-binding helix-turn-helix motif and a dimerization domain (residues 74 -140) that contains two metal binding sites.…”

mentioning

confidence: 99%

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Motion of the DNA-binding Domain with Respect to the Core of the Diphtheria Toxin Repressor (DtxR) Revealed in the Crystal Structures of Apo- and Holo-DtxR

Pohl¹,

Holmes²,

Hól³

1998

Journal of Biological Chemistry

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The diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae is a divalent metal-activated repressor of chromosomal genes that encode proteins responsible for siderophore-mediated iron uptake and also of the gene of certain corynebacteriophages that encodes diphtheria toxin. DtxR consists of two 25.3-kDa three-domain subunits and is a member of a family of related repressor proteins in several Gram-positive bacterial species, some of which are important human pathogens. In this paper, we report on the first high resolution crystal structures of apo-DtxR in two related space groups. In addition, crystal structures of Zn-DtxR were determined in the same two space groups. The resolutions of the structures range from 2.2 to 2.4 Å. The four refined models of the apo-and the holo-repressor exhibit quite similar metal binding centers, which do, however, show higher thermal motion in the apo-structures. All four structures reported differ from each other in one important aspect. The N-terminal DNAbinding domain and the last 20 residues of the dimerization domain of each subunit move significantly with respect to the core of the DtxR dimer, which consists of residues 74 -120 from both subunits. These results provide the first indication of a conformational change that may occur upon binding of the holo-repressor to DNA.

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Section: Table IV Pairwise Comparison Of the Apo-and Zn-dtxr Structuressupporting

confidence: 61%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Table IV Pairwise Comparison Of the Apo-and Zn-dtxr Structuresmentioning

confidence: 99%

mentioning

confidence: 99%

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Motion of the DNA-binding Domain with Respect to the Core of the Diphtheria Toxin Repressor (DtxR) Revealed in the Crystal Structures of Apo- and Holo-DtxR

Pohl¹,

Holmes²,

Hól³

1998

Journal of Biological Chemistry

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A rapid method for positioning small flexible molecules, nucleic acids, and large protein fragments in experimental electron density maps

et al. 1999

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A Monte Carlo procedure, encoded in the program Blob, has been developed and tested for the purpose of positioning large molecular fragments or small flexible molecules in electron density maps. The search performed by the algorithm appears to be sufficiently thorough to accurately position a small flexible ligand in well-defined density while remaining sufficiently random to offer interesting alternate suggestions for density representing disordered binding modes of a ligand. Furthermore, the algorithm is shown to be efficient enough to accurately position large rigid molecular fragments. In the first of the test cases with large molecular fragments, Blob was surprisingly effective in positioning a poly-alanine model of a 53-residue domain in poor electron density resulting from molecular replacement with a partial model. At 3.0 A resolution the domain was positioned consistently within 0.2 A of its experimentally determined position. Even at 6.0 A resolution Blob could consistently position the domain to within 0.75 A of its actual position. A second set of tests with large molecular fragments revealed that Blob could correctly position large molecular fragments with quite significant deviations from the actual structure. In this test case, fragments ranging from a 170-residue protein domain with a 3.8 A rms deviation from the actual structure to a 22-base pair ideal B-form DNA duplex were positioned accurately in a 3.2 A electron density map derived from multiple isomorphous replacement methods. Even when decreasing the quality of the maps, from a figure of merit of 0.57 to as low as 0. 35, Blob could still effectively position the large protein domain and the DNA duplex. Since it is efficient, can handle large molecular fragments, and works in poor and low resolution maps, Blob could be a useful tool for interpreting electron density maps in de novo structure determinations and in molecular replacement studies. Proteins 1999;36:512-525.

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A database method for automated map interpretation in protein crystallography

et al. 1999

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A significant portion of new protein structures contain folds that are related to those seen before. During the development of a computer program that can accurately position, in electron density maps, large protein domains with large structural deviations, it became apparent that the redundancy in protein folds could be used in a non trivial manner during a protein structure determination. As a result a computational procedure, Database Assisted Density Interpretation (DADI), was developed and tested to aid in the building of models in protein crystallography and to assist in interpreting electron density maps. The initial tests of the DADI procedure using a small database of protein domains are described. The philosophy is to first work with entire domains then with the secondary structure elements of these domains and finally with individual residues of the secondary structure elements via Monte Carlo, "chopping" and "clipping" procedures, respectively. The first test case was a traceable 3.2 A multiple isomorphous replacement with anomalous scattering (MIRAS) electron density map of a human topoisomerase I-DNA complex. The second test case uses poor electron density for the third domain of the diphtheria toxin repressor resulting from a molecular replacement solution with the first two domains. Despite the fact that a fairly small database was employed in these test cases, the DADI procedure was able to find a large portion of the protein backbone with very few errors. In the first case nearly 45% of the backbone and more than 80% of the secondary structure was placed automatically. In the second test case nearly 50% of the third domain was automatically detected. A particular encouraging result was that in both cases more than 75% of the beta sheet secondary structure was found automatically by the DADI procedure. Clearly, the procedures employed are promising avenues to exploit the current explosion of protein structures for the determination of future structures. Proteins 1999;36:526-541.

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

Cited by 39 publications

References 24 publications

Motion of the DNA-binding Domain with Respect to the Core of the Diphtheria Toxin Repressor (DtxR) Revealed in the Crystal Structures of Apo- and Holo-DtxR

Motion of the DNA-binding Domain with Respect to the Core of the Diphtheria Toxin Repressor (DtxR) Revealed in the Crystal Structures of Apo- and Holo-DtxR

A rapid method for positioning small flexible molecules, nucleic acids, and large protein fragments in experimental electron density maps

A database method for automated map interpretation in protein crystallography

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