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
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
- 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.
