Sandbox reserved 330
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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 | ||||||||
Overview of YdcEOverview of YdcE
The Bacillus subtilis YdcE gene encodes an endoribonuclease called EndoA, which is a member of the MazF/PemK family of bacterial toxin and the protein encoded by the YdcD gene is an inhibitor of its activity[1]. EndoA cleaves at the UAC sequence, which is predicted to be a single stranded conformation, and has an overlapping cleavage site specificity with the MazF E.coli homologues. EndoA activity results in cleavage products with a 3’phosphate and 5’OH group, which is typical of degradative RNAses that functions in the absence of divalent cations [1].
Background on Toxin and Anti-toxin complexBackground on Toxin and Anti-toxin complex
Addiction modules, consisting of a toxin and antitoxin pair, are controlled by operons which, are autoregulated at the transcriptional level. Bacteria rely on addiction modules to maintain plasmids within populations, and cells that do not inherit the plasmid encoded operon will not produce antitoxin and will be inhibited by the toxin via post segregational killing[1]. Once this operon is expressed, the bacterial strain is addicted to the antitoxin for survival. It is known that genomes of most bacteria have a toxin-antitoxin loci, which have been shown to be induced by stressful conditions[1]. So thus, these modules play an important role in plasmid partitioning and cellular response to stress, where the maintenance of these modules prevents the lethal effect of toxin on cells.
Previous studies of toxin families include MazF, ChpAK, and PemK, which all code for endoribonuclease that activates cellular mRNAs by cleaving them at specific sites. Recently, there is a Bacillus subtilis gene product discovered, EndoA, that is a member of RNAses, which is the gene product of the YdcE gene. This EndoA has similar cleavage pattern specificity as MazF and PemK, with cleavage products of a 3’phosphate and 5’OH group[1]. Further study revealed that a coexpression of an upstream gene, YdcD reverses the effects of the YdcE toxin, and thus, this is the first toxin-antitoxin system identified for Bacillus subtilis. However, further research is necessary to determine the functionality of the toxin-antitoxin complex.
StructureStructure
Crystallization of the YdcE gene product revealed a crystal with space group of P6522, where a= 56.63, b=56.63, and c=138.257. The final structure model of the YdcE protein was determined to be 2.1 angstroms with an R-factor of 15.9%. The YdcE protein consists of 117 amino acids and is approximately 14 kDa[2]. It is a compact single domain alpha/beta protein, with 3 α helices and 7 β strands. Five out of seven beta strands, β1, β2, β3, β6, and β7 forms an antiparallel sheet. While two of the remaining strands, β4, β5, and C terminus (containing Asp115) of the β3 strand forms a smaller sheet[2].
The structure itself is a dimer interface between monomers that is related by a two fold axis, and it exists as a dimer in solution as well. The dimer is a convex surface with a flat surface that includes 3 α helix that has C-terminal tails protruding. The convex surface is an extensive hydrophobic surface between the two monomers, and include Each monomer has a β6 strand that is paired with each other through hydrogen bonds between the amide of the Thr82 and the carbonyl oxygen of Ile 80.
On the convex side of the dimer, hydrogen bonds exist between amides of Ser 19, to the side chain of Asp 84, along with salt bridges between Glu 20 and Arg 87[2]. Between these salt bridges, the Arg 81 of each monomer are buried in the dimer interface and is stabilized by water-mediated hydrogen bonds. Other dimer interactions of the YdcE protein include a hydrogen bond between carbonyl oxygen of Ser 110 and the amide of Asn 32, and between the carbonyl oxygen of Ala 112 and NE of Arg5[2].
The YdcE protein has similar structures to other proteins, such as Kid from E.coli in plasmid R1, and CcdB from E.coli in plasmid F. These similarities include a five stranded antiparallel sheet and a smaller three stranded β-sheet with a C-terminal α helix[2]. YdcE shares 27% sequence similarity with Kid and 7% with CcdB. However, the electronegative surface potential of YdcE is more negative than Kid and CcdB, with a pI of 4.7[3]. This is largely due to having six charged amino acids; Asp 96, Asp 97, Glu 98, Glu 105, Asp 101, and Asp 104[2].
Active SiteActive Site
Complexes of YdcE reveal that the two active sites of the enzyme are located peripherally at the dimer interface, and are shown to be composed of residues contributed from both monomers of the dimer. The two active sites of the native YdcE protein structure have a few differences to those when complexed with other proteins, however, the largest difference is in the repositioning of the aromatic ring of the Phe8, which is rotated approximately by 32° in the complex structure relative to the native YdcE protein[3].
The active site of the YdcE protein is composed of residues from both monomers, with key active site residues consisting of Pro1, Arg 11, Arg 38, Phe50. Dimerization of the two monomers include Pro1, which is presumed to be the catalytic base and is from one subunit, while Phe8, Arg 10, Trp 51, and Tyr72 are from the other monomer[3].
- ↑ 1.0 1.1 1.2 1.3 1.4 Pellegrini, O., Mathy, N., Gogos.A., Shapiro, L., Condon, C. The Bacillus subtilis YdcDE operon encodes an endoribonuclease of the MazF/PemK family and its inhibitor. Molecular Microbiology.2005. June;56(5):1139-1148
- ↑ 2.0 2.1 2.2 2.3 2.4 2.5 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
- ↑ 3.0 3.1 3.2 Almrud, J.J., Kern, A.D., Wang, S.C., Czerwinski, R.M., Johnson, W.H., Murzin, A.G., Hackert, M.L., Whitman, C.P. The crystal structure of YdcE, a 4-oxalocrotonate tautomerase homologue from Escherichia coli., confirms the structural basis for oligomer diversity. Biochemistry.2002. August;41(40):12010-12024