Sandbox Reserved 307: Difference between revisions
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The P<sub>21</sub> structure forms a final complex consisting of six EF-Tu proteins, GDP, tetracycline, Mg<sup>2+</sup>, and 244 water molecules<ref name="gp"/>. The final complex of the P<sub>43212</sub> crystal is composed of one protein copy, GDP, tetracycline, Mg<sup>2+</sup>, SO<sub>2</sub>, glyoxylic acid, 3 Na<sup>+</sup> and 160 H2O molecules <ref name="gp"/>. In either case, the binding site for tetracycline is located in domain one, allowing it to interact with the major functional groups. Residues Tet O11 and Tet O12 of tetracycline co-ordinate Mg<sup>2+</sup><ref name="gp"/>. There is also an interaction with the phosphate of the GDP and the residues Thr25 and Asp80 <ref name="gp"/>. When bound, tetracycline replaces two well ordered water molecules from the EF-Tu structure. | The P<sub>21</sub> structure forms a final complex consisting of six EF-Tu proteins, GDP, tetracycline, Mg<sup>2+</sup>, and 244 water molecules<ref name="gp"/>. The final complex of the P<sub>43212</sub> crystal is composed of one protein copy, GDP, tetracycline, Mg<sup>2+</sup>, SO<sub>2</sub>, glyoxylic acid, 3 Na<sup>+</sup> and 160 H2O molecules <ref name="gp"/>. In either case, the binding site for tetracycline is located in domain one, allowing it to interact with the major functional groups. Residues Tet O11 and Tet O12 of tetracycline co-ordinate Mg<sup>2+</sup><ref name="gp"/>. There is also an interaction with the phosphate of the GDP and the residues Thr25 and Asp80 <ref name="gp"/>. When bound, tetracycline replaces two well ordered water molecules from the EF-Tu structure. | ||
<applet load='2hdn' size='275' color='color' frame='true' align='left' scene='Sandbox_307/> | |||
There are three domains making up the EF-Tu molecule. <scene name='Sandbox_Reserved_307/Domain_one/1'>Domain 1</scene> <ref name="gp"/>, visualized in green, is the guanine-nucleotide domain made from residues 8-40 and 59-204. <scene name='Sandbox_Reserved_307/Domain_2/1'>Domain 2</scene>, purple, is composed on residues 205-298 and <scene name='Sandbox_Reserved_307/Domain_3/1'>Domain 3</scene> <ref name="gp"/>, yellow, of residues 299-393. The fragments 8-44 and 59-393 are associated through non-covalent interactions and retain their native conformation, with the exception of residues 40-44 and 260-263 <ref name="gp"/>. | There are three domains making up the EF-Tu molecule. <scene name='Sandbox_Reserved_307/Domain_one/1'>Domain 1</scene> <ref name="gp"/>, visualized in green, is the guanine-nucleotide domain made from residues 8-40 and 59-204. <scene name='Sandbox_Reserved_307/Domain_2/1'>Domain 2</scene>, purple, is composed on residues 205-298 and <scene name='Sandbox_Reserved_307/Domain_3/1'>Domain 3</scene> <ref name="gp"/>, yellow, of residues 299-393. The fragments 8-44 and 59-393 are associated through non-covalent interactions and retain their native conformation, with the exception of residues 40-44 and 260-263 <ref name="gp"/>. | ||
In domain 1 of EF-Tu, the protein-protein interactions involved four anti-parallel β-sheet hydrogen bonds between residues 64-66’ and 66-64’ (where the primes indicate non-crystallographically related residues)<ref name="gp"/>. Hydrogen bonds are also formed between the side chain of Asn63 and Glu68 as well as the main chain residues Ile62 with His66 and Glu60 <ref name="gp"/>. | In domain 1 of EF-Tu, the protein-protein interactions involved four anti-parallel β-sheet hydrogen bonds between residues 64-66’ and 66-64’ (where the primes indicate non-crystallographically related residues)<ref name="gp"/>. Hydrogen bonds are also formed between the side chain of Asn63 and Glu68 as well as the main chain residues Ile62 with His66 and Glu60 <ref name="gp"/>. | ||
In the second domain, residues 216,259,261,262, and 287 form two electrostatic interactions and six hydrogen bonds form with 216’, 261’, 262’, and 283’ to create a mobile loop from Arg262-Leu264 <ref name="gp"/>. There are also stabilizing van der Waals interactions between the dimers. The tetracycline rings are nearly parallel to the dimer unit creating a hydrophobic environment. | In the second domain, residues 216,259,261,262, and 287 form two electrostatic interactions and six hydrogen bonds form with 216’, 261’, 262’, and 283’ to create a mobile loop from Arg262-Leu264 <ref name="gp"/>. There are also stabilizing van der Waals interactions between the dimers. The tetracycline rings are nearly parallel to the dimer unit creating a hydrophobic environment. | ||
Each dimer of the Tm-EF-Tu-MgGDP complex is related to another pair by a pseudo-twofold axis with minimal intermolecular interactions <ref name="gp"/>. Conformations of the protein complexed | Each dimer of the Tm-EF-Tu-MgGDP complex is related to another pair by a pseudo-twofold axis with minimal intermolecular interactions <ref name="gp"/>. Conformations of the protein complexed with tetracycline do not appear to differ from those which are non-complexed, suggesting little effect on the proteins structure <ref name="gp"/>. | ||
<scene name='Sandbox_Reserved_307/Removal/1'>Residues 40-44 and 260-263</scene> are disordered when EF-Tu-MgGDP undergoes trypsin modification <ref name="gp"/>. In addition, residues 1-7 and 45-58 are removed, the latter of which makes up the Switch I loop <ref name="gp"/>. The Switch I loop conformation is regulated by the binding of MgGDP and MgGTP. The presence of tetracycline may affect the conversion rate between the EF-Tu-MgGTP and EF-Tu-MgGDP conformations, affecting the interaction with the ribosome <ref name="gp"/>. In this way, tetracycline may act to inhibit protein synthesis by preventing the binding of aminoacyl-tRNA to the A site of the ribosome. | |||
==Function== | ==Function== | ||
EF-Tu binds an aminoacylated tRNA molecule in the cytoplasm and allows entry into the ribosome <ref name="gp"/>. | |||
EF-Tu binds an aminoacylated tRNA molecule in the cytoplasm and allows entry into the ribosome <ref name="gp"/>. EF-Tu binds a cavity between the 30S and 50S ribosomal subunits, while the tRNA anticodon associates with the mRNA codon in the A site of the ribosome <ref name="gp3">PMID: 19536129 </ref>. If the pairing is incorrect, the tRNA will likely leave the ribosome. However, if a correct pairing has occurred, EF-Tu hydrolyzes guanosine triphosphate (GTP) to the diphosphate form (GDP)<ref name="gp"/>. This hydrolysis results in a change in conformation, releasing the tRNA into the ribosome. The dissociation allows the tRNA molecule to then completely interact with the A site of the ribosome. Then the amino acid on the tRNA can be transferred to the growing peptide chain via covalent bond formation <ref name="gp"/>. The GDP-bound EF-Tu molecule can then interact with EF-Ts, which exchanges the GDP for a new GTP and prepares EF-Tu to interact with another charged amino acid, once EF-G has acted for translocation along the mRNA sequence. In this way, EF-Tu is a vital component in the process of lengthening a peptide during protein synthesis.[[Image:EF-TUFigure.jpg | frame="true" | caption='EF-Tu and EF-G cycle during protein synthesis at the ribosome']] | |||
EF-Tu has also been shown to play a role in the inhibition of tetracycline during protein synthesis in many different organisms <ref name="gp"/>. EF-Tu may be a target protein of tetracycline, although this idea has commonly been dismissed because the ribosome may be inhibited in the presence of the antibiotic <ref name="gp"/>. Now however, it has been demonstated that Tm-EF-Tu-MgGDP is bound in a complex during crystallization, binding the the GTPase active site. This information could be useful for developing mechanisms of counteracting resistance to this particular antibiotic <ref name="gp"/>. | EF-Tu has also been shown to play a role in the inhibition of tetracycline during protein synthesis in many different organisms <ref name="gp"/>. EF-Tu may be a target protein of tetracycline, although this idea has commonly been dismissed because the ribosome may be inhibited in the presence of the antibiotic <ref name="gp"/>. Now however, it has been demonstated that Tm-EF-Tu-MgGDP is bound in a complex during crystallization, binding the the GTPase active site. This information could be useful for developing mechanisms of counteracting resistance to this particular antibiotic <ref name="gp"/>. | ||
==References== | ==References== | ||
<references/> | <references/> | ||