Sandbox reserved 330
From Proteopedia
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| 1ne8, resolution 2.10Å () | |||||||||
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| Ligands: | , | ||||||||
| Gene: | ydcE (Bacillus subtilis) | ||||||||
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| Resources: | FirstGlance, OCA, RCSB, PDBsum, TOPSAN | ||||||||
| Coordinates: | save as pdb, mmCIF, xml | ||||||||
Background Information
- addition modules have two genes: toxin genes and an antitoxin genes - modules mediate plasmid maintenance by killing plasmid free cells via what you call, post segregational killing. - Operons are autoregulated at transcriptional level to control toxin/antitoxin modules - Maintenance prevents the lethal effect of the toxin. - There have been two structures that are found, that have similar 3D structure - 1. Kid from E.coli in plasmid R1 - 2. CcdB from E.coli in plasmid F. - modules found so far such as ChpA/MazEF and ChpB, which are homologous genes to the addiction modules, are involved in cell death that is thought to be triggered by antibiotics.
- operons encode stable toxin and their antidote - opersons play an important role in plasmid partitioning and cellular response to stress. - Family of toxins (MazF/ChpAK/PemK) encodes endoribonuclease that activates cellular mRNAs by cleaving them at specific sites - This study, there is a B.subtilis gene encode a member of RNases that they call Endo A. - A coexpression of an upstream gene (YdcD) reverses toxin. - YdcD inhibits EndoA activity. - endoA has similar cleavage patterns specificity to MazF/PemK, with cleavage produces with a 3’P and 5’OH group. - This is the first example of an antitoxin-toxin system of B. subtilis.
- bacteria rely on addiction modules to maintain plasmids within populations. - These modules consist of operon where one cistron encodes a stable toxin and is preceded by an encoding unstable antitoxin. - Cells that do not inherit the plasmid encoded operon will not produce antitoxin and inhibited by the toxin via post segregational killing. - So once this operon is expressed, the bacterial strain is addicted to the antitoxin for survival. - It is known that genomes of most bacteria has a toxin-antitoxin TA loci. - These loci have been shown to be induced by stressful conditions. - It has been shown that antitoxin can also inhibit its own expression by binding to its own promoter region with complexed toxin as corepressor. -
Structure
- sequences of chromosomally encoded protein homologous to MazF toxin of E.coli are part of a group called Cluster of Orthologous Groups of Proteins (COG 2337) - COG 2337 include representation from B. subtilis that do not function as inhibitors. - the first of this group is YdcE 3D structure. - Amplification of YdcE gene with primers, incorporating 5’BamHI and 3’HindIII into pSMT3 expression vector that has 6His-sumo-N-terminal tag. - Purification via affinity and gel filtration. - N-terminal tag is removed. - Crystallized via hanging drop vapour diffusion. - It was found that the crystal space group: P6522 with a= 56.63, b=56.63, and c= 138.257 with a protein molecular per asymmetric unit. - The structure of the YdcE protein was determined using phases derived from three-wavelength multiwavelength anomalous diffraction on single crystal of Se-MET-substituted protein. - The final model of the protein was determined to be 2.1 A with R-factor of 15.9% - There is 117 AA in the YdcE protein - It is a compact single domain alpha/beta protein= 3 alpha helices and 7 beta strands - 5/7 strands= B1,2,3,6,7 forms an antiparallel sheet - 2/7 strands= B4 and B5 and C terminus of B3 forms a smaller sheet. - The structure itself is a dimmer interface between monomers that is related by a two fold axis. - Light scattering and gel filtration reveal that YdcE is a dimer in solution too. - The dimer itself is a convex surface. - Capped loops are located btw strands B1 and B2 - Flat surface that includes the 3 alpha helix that has C-terminal tails protruding. - The convex surface is an extensive hydrophobic surface between two monomers. - This hydrophobic surface includes Ile 30, Ile 43, Ile 111, Leu 107, Ile 80 and Ile 114. - Each monomer has B6 strand that pair with each other through H bonds between the amide of Thr 82 and the carbonyl oxygen of Ile80. - On the convex side of the dimer, H bonds between amides of Ser 19 to the side chain of Asp 84, also there are salt bridges between Glu 20 and Arg 87. - Between these salt bridges, the Arg 81 of each monomer are buried in the dimer interface and is stabilized by water mediated H bonds. - There are other dimer interactions which include. - 1. H bond between carbonyl oxygen of Ser 110 and the amide of Asn 32 - 2. Carbonyl oxygen of Ala 112 and Ne(NE) of Arg 5.
- YdcE, Kid and CcdB all share similar folds: 5 stranded antiparallel sheet, smaller 3 stranded B-sheet with C-terminal alpha helix. - YdcE shares 27% sequence similarity with Kid and 7% with CcdB. - The C-terminal helix is on the flat side of YdcE protein. - The surface potential is more negative than Kid and CcdB though. - YdcE has 6 charged AA - 1. Asp 96 - 2. Asp 97 - 3. Glu 98 - 4. Glu 105 - 5. Asp 101 - 6. Asp 104 Image:Image for proto1.png
- the protein surface and C-terminus of YdcE protein is involved in toxin interaction with it’s target.
- The C-terminus of these homologs vary, so variability may reflect substrate specificity within the protein family.
[1]
Function
- ↑ Gogos A, Mu H, Bahna F, Gomez CA, Shapiro L. Crystal structure of YdcE protein from Bacillus subtilis. Proteins. 2003 Nov 1;53(2):320-2. PMID:14517982 doi:http://dx.doi.org/10.1002/prot.10457
