Sandbox 201
From Proteopedia
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:Metal ions are essential for the nucleotidyltransferase catalysis by all T4 DNA and T4 RNA ligases, which use the same two-metal ions mechanism. | :Metal ions are essential for the nucleotidyltransferase catalysis by all T4 DNA and T4 RNA ligases, which use the same two-metal ions mechanism. | ||
| - | :The enzyme binds <scene name='Sandbox_201/Mg/1'>two magnesium ions</scene>. The true substrate in the adenylation reaction is the ATP-Mg<sup>2+</sup> complex <ref>Cherepanov, A. V., and de Vries, S. (2002) J. Biol. Chem. 277, 1695–1704</ref>, but nucleotidyltransferase enzymes cannot bind ATP-Mg<sub>2</sub> directly. They bind ATP-Mg first, then a second Mg<sup>2+</sup> ion. Each oh these cations <scene name='Sandbox_201/Mg_apc_residues/1'>interact via hydrogen bonds</scene> with one phosphoryl oxygen from AMPcPP, three water molecules and two residues (Gly269 and Asp272), which both belong to the C-terminal domain. | + | :The enzyme binds <scene name='Sandbox_201/Mg/1'>two magnesium ions</scene> Mg<sup>2+</sup>. The true substrate in the adenylation reaction is the ATP-Mg<sup>2+</sup> complex <ref>Cherepanov, A. V., and de Vries, S. (2002) J. Biol. Chem. 277, 1695–1704</ref>, but nucleotidyltransferase enzymes cannot bind ATP-Mg<sub>2</sub> directly. They bind ATP-Mg first, then a second Mg<sup>2+</sup> ion. Each oh these cations <scene name='Sandbox_201/Mg_apc_residues/1'>interact via hydrogen bonds</scene> with one phosphoryl oxygen from AMPcPP, three water molecules and two residues (Gly269 and Asp272), which both belong to the C-terminal domain. |
| - | :The enzyme binds <scene name='Sandbox_201/Ca/1'>four calcium ions</scene>. <scene name='Sandbox_201/Ca6_apc_residues/1'>Two</scene> are coordinated to six water molecules. They do not directly interact with the enzyme, but via water molecules interacting with Glu227, Glu159, Lys99, Glu100, and Tyr246 via hydrogen bonds. They also interact with one phosphoryl oxygen of the AMPcPP. <scene name='Sandbox_201/Ca4_residues/2'>Two other</scene> are coordinated to four water molecules and interact with three enzyme residues ( | + | :The enzyme binds <scene name='Sandbox_201/Ca/1'>four calcium ions</scene> Ca<sup>2+</sup>. <scene name='Sandbox_201/Ca6_apc_residues/1'>Two</scene> are coordinated to six water molecules. They do not directly interact with the enzyme, but via water molecules interacting with Glu227, Glu159, Lys99, Glu100, and Tyr246 via hydrogen bonds. They also interact with one phosphoryl oxygen of the AMPcPP. <scene name='Sandbox_201/Ca4_residues/2'>Two other</scene> are coordinated to four water molecules and interact with three enzyme residues (Ile211 and Asp212) via hydrogen bonds. |
| + | :Calcium are very important in enzyme Rnl1 structural biology, because the enzyme cristallises only with Ca<sup>2+</sup>. | ||
* RNA binding site | * RNA binding site | ||
| - | :The RNA-Rnl1 complex have not be crystallized yet, because the enzyme seems to crystallize only with | + | :The RNA-Rnl1 complex have not be crystallized yet, because the enzyme seems to crystallize only with AMPcPP, which is incompatible with the presence of RNA in the active site. That's why we are not able to define precisely the RNA binding site. But there are some elements tending to indicate of a possible RNA biding site in the <scene name='Sandbox_201/C-terminal_domain_chainb/1'>C-terminal domain</scene> : |
::- The analysis of charge distribution on the protein surface shows that the Rnl1 surface is negatively charged, apart from the C-terminal domain. The positive charges present on this domain could interact with the polyanion-like RNA backbone. | ::- The analysis of charge distribution on the protein surface shows that the Rnl1 surface is negatively charged, apart from the C-terminal domain. The positive charges present on this domain could interact with the polyanion-like RNA backbone. | ||
::- The RNA have to be close to the ATP binding site to enable the AMP transfer from Lys99. The RNA could bind at the surface of the C-terminal domain, allowing the anticodon loop to be positionned toward the ATP binding site. | ::- The RNA have to be close to the ATP binding site to enable the AMP transfer from Lys99. The RNA could bind at the surface of the C-terminal domain, allowing the anticodon loop to be positionned toward the ATP binding site. | ||
::- Moreover, the C-terminal helical structure matches the tRNA structure. | ::- Moreover, the C-terminal helical structure matches the tRNA structure. | ||
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| + | * AMPcPP binding site | ||
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| + | AMPcPP does not allow the nucleotidyltransferase function of the enzyme, but it seems necessary to Rnl1 cristallyzation. | ||
</StructureSection> | </StructureSection> | ||
Revision as of 21:04, 2 January 2012
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| 2c5u, resolution 2.21Å () | |||||||||
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| Ligands: | , , , | ||||||||
| Non-Standard Residues: | |||||||||
| Activity: | RNA ligase (ATP), with EC number 6.5.1.3 | ||||||||
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| Resources: | FirstGlance, OCA, PDBsum, RCSB | ||||||||
| Coordinates: | save as pdb, mmCIF, xml | ||||||||
T4 RNA ligase (Rnl1) catalyzes the formation of phosphodiester bonds between the 5'-phosphate and the 3'-hydroxyl termini of single-stranded nucleic acids. T4 RNA ligase is a member of a distinct subgroup of RNA ligases along with a fungal tRNA ligase(Trl1), a putative baculovirus RNA ligase and RNA ligase from the bacteriophages RM378 and TS2126. Rnl1 is also the first RNA ligase whose complete crystal structure was determined. Rnl1 is in fact a tRNA repair enzyme used by the T4 bacteriophage to escape hosts antiviral response. Enzyme functioning requires ATP and divalent metal ions. The T4 ligase repairs the tRNALys by joining its 5'-PO4 and 3'-OH groups via series of three nucleotidyl transfer steps in a ping-pong enzymatic mechanism. First, the Lys99 of the enzyme reacts with the a phosphorus of ATP and forms a covalent intermediate: ligase-(lysyl-N)-AMP. Pyrophosphate is also produced during this step. Secondly, AMP is transferred from the intermediate to the 5'- PO4 terminus of a tRNA to form an tRNA-adenylate intermediate (AppRNA). Finally, the ligase catalyzes the attack of the 3'-OH terminus of the tRNA on the tRNA-adenylate and the two termini are joined via a phosphodiester bond, the AMP is released.
Contents |
Biological role
The biological role of Rnl1 is to repair a break in the anticodon loop of E.coli tRNALys and in this way to evade bacteria host antiviral defense mechanism invoked following phage infection. Bacteria have a tRNALys-specific anticodon nuclease (ACNase) which is normally kept latent by association of its core protein, PrrC, with the endonuclease EcoprrI. Upon infection, the bacteriophage expresses a T4 Stp peptide, which inhibits EcoprrI. EcoprrI dissociates from PrrC and the ACNase becomes active. The anticodon nuclease then cleaves the anticodon loop of the tRNALys which blocks phage protein synthesis and, as a consequence, stops the infection. Bacteriophage T4 has developed way to overcome this defense mechanism using the tRNA ligase and a polynucleotide kinase (PnK) to repair the in the tRNA anticodon loop. T4 Rnl1 and T4 polynucleotide kinase–phosphatase (PnK) together form a two-component repair system that repairs the tRNA break made by the host anticodon nuclease. First, PnK remodels the ends of the broken tRNA by converting the 2',3' cyclic phosphate to a 3'-OH, 2'-OH and by phosphorylating the 5'-OH end to form a 5'-PO4. Rnl1 then joins the 3'-OH and 5'- PO4 RNA ends to form a standard 3'–5' phosphodiester bond.
Structure
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External Resources
References
- ↑ Cherepanov, A. V., and de Vries, S. (2002) J. Biol. Chem. 277, 1695–1704
