PMID: 17057344 </ref>. The resulting complex, Tm-EF-Tu-MgGDP, is composed of three domains and can be found in two crystalline structures.
Structure
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, six copies of EF-Tu, GDP, tetracycline, and Mg2+ 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.
There are three domains making up the EF-Tu molecule. Domain 1 is the guanine-nucleotide domain made from residues 8-40 and 59-204 [1]. Domain 2 is composed on residues 205-298 and the final domain of residues 299-393 [1]. 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 to tetracycline to not appear to differ from those which are non-complexed, suggesting little effect on the proteins structure [1].
Residues 40-44 and 260-263 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 that 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.
Function
EF-Tu binds an aminoacylated tRNA molecule and allows entry into the ribosome [1]. The tRNA anticodon associates with the mRNA codon in the A site of the ribosome. 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 of the tRNA, which allows it to dissociate from EF-Tu. The dissociation allows the tRNA molecule to then completely interact with the A site of the ribosome the tRNA can be transferred to the growing peptide chain via covalent bond formation [1]. In this way, EF-Tu is a vital component in the propcess 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 [1].
References
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