Crystal structure of CcdB, a topoisomerase poison from E. coli 1 1Edited by T. Richmond

Loris, Remy; Dao‐Thi, Minh‐Hoa; Bahassi, El Mustapha; Melderen, Laurence Van; Poortmans, F.; Liddington, Robert; Couturier, Martine; Wyns, Lode

doi:10.1006/jmbi.1998.2395

Cited by 140 publications

(145 citation statements)

References 36 publications

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“…At eight of 10 positions, Asp substitution leads to protein inactivation (Table 1). While this work was in progress, the crystal structure of CcdB was solved (30). In agreement with the mutational data, eight of 10 predicted buried residues are Ͼ90% buried, whereas Val-53 and Leu-96 are 80% and 75% buried, respectively.…”

Section: Resultssupporting

confidence: 65%

Design of temperature-sensitive mutants solely from amino acid sequence

Chakshusmathi

Mondal

Lakshmi

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Temperature-sensitive (Ts) mutants are a powerful tool with which to study gene function in vivo. Ts mutants are typically generated by random mutagenesis followed by laborious screening procedures. By using the Escherichia coli cytotoxin CcdB as a model system, simple procedures for generating Ts mutants at high frequency through site-directed mutagenesis were developed. Putative buried, hydrophobic residues are selected through analysis of the protein sequence. Residue burial is confirmed by ensuring that substitution of the residue by Asp leads to protein inactivation. At such sites, a Ts phenotype can typically be generated either by (i) substitution of two predicted, buried residues with the 18 remaining amino acids or (ii) introduction of Lys, Ser, Ala, and Trp at three to four predicted buried sites. By using these design strategies, 17 tight Ts mutants of CcdB were isolated at four predicted buried sites. The rules were further verified by making several Ts mutants of yeast Gal4 at residues 68, 69, and 70. No Ts mutants of either protein have been previously reported. Such Ts mutants of Gal4 can be used for conditional expression of a variety of genes by using the well characterized upstream-activatingsequence-Gal4 system.

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Section: Resultssupporting

confidence: 65%

Design of temperature-sensitive mutants solely from amino acid sequence

Chakshusmathi

Mondal

Lakshmi

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…2 The first toxin structure to be determined was the E. coli F plasmid CcdB toxin. 3 The structures of many toxins, antitoxins, and TA complexes have been determined since, including E. coli plasmid R1 Kid toxin, 4 E. coli MazE-MazF TA complex, 5 broad-hostrange low-copy-number plasmid pSM19035 from Streptococcus pyogenes e 2 f 2 TA complex, 6 E. coli YoeB toxin and YefM2-YoeB TA complex, 7 archaeal Pyrococcus horikoshii aRelE-aRelB TA complex, 8 Neisseria gonorrhoeae FitAB TA complex, 9 solution structure of the broad host range low-copy-number plasmid pRK2/RP4 ParD antitoxin, 10 Mycobacterium tuberculosis (Mtb)…”

Section: Introductionmentioning

confidence: 99%

The crystal structure of the Rv0301‐Rv0300 VapBC‐3 toxin—antitoxin complex from M. tuberculosis reveals a Mg²⁺ ion in the active site and a putative RNA‐binding site

et al. 2012

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VapBC pairs account for 45 out of 88 identified toxin-antitoxin (TA) pairs in the Mycobacterium tuberculosis (Mtb) H37Rv genome. A working model suggests that under times of stress, antitoxin molecules are degraded, releasing the toxins to slow the metabolism of the cell, which in the case of VapC toxins is via their RNase activity. Otherwise the TA pairs remain bound to their promoters, autoinhibiting transcription. The crystal structure of Rv0301-Rv0300, an Mtb VapBC TA complex determined at 1.49 Å resolution, suggests a mechanism for these three functions: RNase activity, its inhibition by antitoxin, and its ability to bind promoter DNA. The Rv0301 toxin consists of a core of five parallel beta strands flanked by alpha helices. Three proximal aspartates coordinate a Mg 21 ion forming the putative RNase active site. The Rv0300 antitoxin monomer is extended in structure, consisting of an N-terminal beta strand followed by four helices. The last two helices wrap around the toxin and terminate near the putative RNase active site, but with different conformations. In one conformation, the C-terminal arginine interferes with Mg 21 ion coordination, suggesting a mechanism by which the antitoxin can inhibit toxin activity. At the N-terminus of the antitoxin, two pairs of Ribbon-Helix-Helix (RHH) motifs are related by crystallographic twofold symmetry. The resulting hetero-octameric complex is similar to the FitAB system, but the two RHH motifs are about 30 Å closer together in the Rv0301-Rv0300 complex, suggesting either a different span of the DNA recognition sequence or a conformational change.

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“…The protein is an inhibitor of DNA gyrase and is a potent cytotoxin in Escherichia coli (9). Crystallographic structures of CcdB in the free and gyrase bound forms (10,11) are also available. Transformation of normal E. coli cells with plasmid expressing the wild-type (WT) ccdb gene results in cell death.…”

mentioning

confidence: 99%

Mutagenesis-based definitions and probes of residue burial in proteins

Bajaj

Chakrabarti²,

Varadarajan³

2005

Proc. Natl. Acad. Sci. U.S.A.

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Every residue of the 101-aa Escherichia coli toxin CcdB was substituted with Ala, Asp, Glu, Lys, and Arg by using site-directed mutagenesis. The activity of each mutant in vivo was characterized as a function of Controller of Cell Division or Death B protein (CcdB) transcriptional level. The mutation data suggest that an accessibility value of 5% is an appropriate cutoff for definition of buried residues. At all buried positions, introduction of Asp results in an inactive phenotype at all CcdB transcriptional levels. The average amount of destabilization upon substitution at buried positions decreases in the order Asp>Glu>Lys>Arg>Ala. Asp substitutions at buried sites in two other proteins, maltose-binding protein and thioredoxin, also were shown to be severely destabilizing. Ala and Asp scanning mutagenesis, in combination with dose-dependent expression phenotypes, was shown to yield important information on protein structure and activity. These results also suggest that such scanning mutagenesis data can be used to rank order sequence alignments and their corresponding homology models, as well as to distinguish between correct and incorrect structural alignments. With continuous reductions in oligonucleotide costs and increasingly efficient site-directed mutagenesis procedures, comprehensive scanning mutagenesis experiments for small proteins͞domains are quite feasible.accessibility ͉ aspartate ͉ scanning mutagenesis ͉ phenotype

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Crystal structure of CcdB, a topoisomerase poison from E. coli 1 1Edited by T. Richmond

Cited by 140 publications

References 36 publications

Design of temperature-sensitive mutants solely from amino acid sequence

Design of temperature-sensitive mutants solely from amino acid sequence

The crystal structure of the Rv0301‐Rv0300 VapBC‐3 toxin—antitoxin complex from M. tuberculosis reveals a Mg²⁺ ion in the active site and a putative RNA‐binding site

Mutagenesis-based definitions and probes of residue burial in proteins

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Crystal structure of CcdB, a topoisomerase poison from E. coli 1 1Edited by T. Richmond

Cited by 140 publications

References 36 publications

Design of temperature-sensitive mutants solely from amino acid sequence

Design of temperature-sensitive mutants solely from amino acid sequence

The crystal structure of the Rv0301‐Rv0300 VapBC‐3 toxin—antitoxin complex from M. tuberculosis reveals a Mg2+ ion in the active site and a putative RNA‐binding site

Mutagenesis-based definitions and probes of residue burial in proteins

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The crystal structure of the Rv0301‐Rv0300 VapBC‐3 toxin—antitoxin complex from M. tuberculosis reveals a Mg²⁺ ion in the active site and a putative RNA‐binding site