Sandbox reserved 330: Difference between revisions

==Overview 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<ref name="Pellegrini"/>. 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 <ref name="Pellegrini"> 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</ref>.

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== Background on Toxin and Anti-toxin pair==

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<ref name="Pellegrini"/>. 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<ref name="Pellegrini"/>. 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<ref name="Pellegrini"/>. Further study revealed that a coexpression of an upstream gene, YdcD reverses the effects of the YdcE toxin, and thus, this is the first antitoxin-toxin system identified for Bacillus subtilis.  

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 <scene name='Sandbox_reserved_330/Hydrophobic_residues/1'>Ile 30, Ile 43, Ile 111, Leu 107, Ile 80 and Ile 114.</scene> 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<ref name="Gogos"/>.  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<ref name="Gogos"/>.  

  [[Image:6_charged_AA.jpg | thumb | right | 400px |6 charged amino acids contributing to electronegative surface potential]]   

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<ref name="Almrud"/>.  

      

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<ref name="Almrud"/>.

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