Sandbox 201: Difference between revisions

Line 57:

: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> Mg2+. The true substrate in the adenylation reaction is the ATP-Mg2+ complex, <ref>Cherepanov, A. V., and de Vries, S. (2002) J. Biol. Chem. 277, 1695–1704</ref> but nucleotidyltransferase enzymes cannot bind ATP-Mg2 directly. They bind ATP-Mg first, then a second Mg2+ ion. Each oh these cations <scene name='Sandbox_201/Mg_apc_residues/2'>~~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> Mg2+. The true substrate in the adenylation reaction is the ATP-Mg2+ complex, <ref>Cherepanov, A. V., and de Vries, S. (2002) J. Biol. Chem. 277, 1695–1704</ref> but nucleotidyltransferase enzymes cannot bind ATP-Mg2 directly. They bind ATP-Mg first, then a second Mg2+ ion. Each oh these cations <scene name='Sandbox_201/Mg_apc_residues/2'>interacts 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> Ca2+. <scene name='Sandbox_201/Ca6_apc_residues/2'>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/3'>Two other</scene> are coordinated to four water molecules and interact with three enzyme residues (Ile211 and Asp212) via hydrogen bonds.

Line 65:

* RNA binding site

: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 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/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 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.

Sandbox 201: Difference between revisions

Navigation menu

Page actions

Page actions

Personal tools

Navigation

Search

Tools

@@ Line 57: / Line 57: @@
 :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> 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/2'>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/2'>interacts 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> Ca<sup>2+</sup>. <scene name='Sandbox_201/Ca6_apc_residues/2'>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/3'>Two other</scene> are coordinated to four water molecules and interact with three enzyme residues (Ile211 and Asp212) via hydrogen bonds.
@@ Line 65: / Line 65: @@
 * RNA binding site
-: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 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/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 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.