Sandbox Reserved 307
| This Sandbox is Reserved from January 10, 2010, through April 10, 2011 for use in BCMB 307-Proteins course taught by Andrea Gorrell at the University of Northern British Columbia, Prince George, BC, Canada. |
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Trypsin-modified Elongation Factor Tu (EF-Tu)Trypsin-modified Elongation Factor Tu (EF-Tu)
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| 2hdn, resolution 2.80Å () | |||||||||
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| Ligands: | , , | ||||||||
| Related: | 2hcj | ||||||||
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| Resources: | FirstGlance, OCA, PDBsum, RCSB | ||||||||
| Coordinates: | save as pdb, mmCIF, xml | ||||||||
General InformationGeneral Information
Trypsin-modified Elongation Factor thermo unstable (Tm-EF-Tu) is a prokaryotic transcription factor and GTPase, which regulates the movement of aminoacyl tRNA into a free site of a ribosome [1]. This protein has been studied in Escherichia coli (E. coli) and shows modification at three regions of the native peptide sequence; Arg-44, Arg-58, and Lys-56 [2]. Tm-EF-Tu tends to form a complex with the antibiotic tetracycline The resulting complex, Tm-EF-Tu-MgGDP, is composed of three domains and can be found in two crystalline structures [1].
StructureStructure
EF-Tu is a 12 chain structure in E. coli that tends to form a complex with the antibiotic tetracycline. Two different crystalline forms of the structure have been identified [1]. In the first crystal form, P21, are arranged as dimers in an asymmetric manner [1]. An alternative form, P43212, is a monomeric, asymmetric unit with tetracycline complexes within the structure [1].
The P21 structure forms a final complex consisting of six EF-Tu proteins, GDP, tetracycline, Mg2+, and 244 water molecules[1]. The final complex of the P43212 crystal is composed of one protein copy, GDP, tetracycline, Mg2+, SO2, glyoxylic acid, 3 Na+ and 160 H2O molecules [1]. 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 Mg2+[1]. There is also an interaction with the phosphate of the GDP and the residues Thr25 and Asp80 [1]. When bound, tetracycline replaces two well ordered water molecules from the EF-Tu structure.
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There are three domains making up the EF-Tu molecule. [1], visualized in green, is the guanine-nucleotide domain made from residues 8-40 and 59-204. , purple, is composed on residues 205-298 and [1], 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 [1].
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)[1]. 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 [1].
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 [1]. 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 [1]. 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 [1].
are disordered when EF-Tu-MgGDP undergoes trypsin modification [1]. In addition, residues 1-7 and 45-58 are removed, the latter of which makes up the Switch I loop [1]. 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 [1]. In this way, tetracycline may act to inhibit protein synthesis by preventing the binding of aminoacyl-tRNA to the A site of the ribosome.
FunctionFunction
EF-Tu binds an aminoacylated tRNA molecule in the cytoplasm and allows entry into the ribosome [1]. 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 [3]. 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)[1]. 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 [1]. 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.
EF-Tu has also been shown to play a role in the inhibition of tetracycline during protein synthesis in many different organisms [1]. 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 [1]. 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 [1].
ReferencesReferences
- ↑ 1.00 1.01 1.02 1.03 1.04 1.05 1.06 1.07 1.08 1.09 1.10 1.11 1.12 1.13 1.14 1.15 1.16 1.17 1.18 1.19 1.20 1.21 1.22 1.23 1.24 1.25 Heffron SE, Mui S, Aorora A, Abel K, Bergmann E, Jurnak F. Molecular complementarity between tetracycline and the GTPase active site of elongation factor Tu. Acta Crystallogr D Biol Crystallogr. 2006 Nov;62(Pt 11):1392-400. Epub, 2006 Oct 18. PMID:17057344 doi:http://dx.doi.org/10.1107/S0907444906035426
- ↑ Wittinghofer A, Frank R, Leberman R. Composition and properties of trypsin-cleaved elongation factor Tu. Eur J Biochem. 1980 Jul;108(2):423-31. PMID:6157532
- ↑ Ticu C, Nechifor R, Nguyen B, Desrosiers M, Wilson KS. Conformational changes in switch I of EF-G drive its directional cycling on and off the ribosome. EMBO J. 2009 Jul 22;28(14):2053-65. doi: 10.1038/emboj.2009.169. Epub 2009 Jun, 18. PMID:19536129 doi:http://dx.doi.org/10.1038/emboj.2009.169