Biophysical characterization and ligand-binding properties of the elongation factor Tu from Mycobacterium tuberculosis

• 来源期刊: 生物化学与生物物理学报（英文版）
• 关键词: tuberculosis MtbEF-Tu protein biosynthesis protein expression and purification protein-guanine nucleotide interaction
• 年，卷（期）: 2019,（2）
• 页码范围: 139-149
• 页数: 11页
• 语种: 英文
• DOI: 10.1093/abbs/gmy164

摘要

Mycobacterium tuberculosis (Mtb) is the key devastating bacterial pathogen responsible for tuberculosis.Increasing emergence of multi-drug-resistant,extensively drug-resistant,and rifampicin/isoniazid-resistant strains of Mtb makes the discovery of validated drug targets an urgent priority.As a vital translational component of the protein biosynthesis system,elongation factor Tu (EF-Tu) is an important molecular switch responsible for selection and binding of the cognate aminoacyltRNA to the acceptor site on the ribosome.In addition,EF-Tu from Mtb (MtbEF-Tu) is involved in the initial step of trans-translation which is an effective system for rescuing the stalled ribosomes from non-stop translation complexes under stress conditions.Given its crucial role in protein biosynthesis,EF-Tu is identified as an excellent molecular target for drug design.Here,we reported the recombinant expression,purification,biophysical characterization,and structural modeling of the MtbEF-Tu protein.Our results demonstrated that prokaryotic expression plasmids of pET28a-MtbEF-Tu could be expressed efficiently in Escherichia coli.We successfully purified the 6x Histagged proteins with a yield of 16.8 mg from 1 I of Luria Bertani medium.Dynamic light scattering experiments showed that MtbEF-Tu existed in a monomeric form,and circular dichroism experiments indicated that MtbEF-Tu was well structured.Moreover,isothermal titration calorimetry experiments displayed that the purified MtbEF-Tu protein possessed intermediate binding affinities for guanosine-5'-triphosphate (GTP) and GDP.The GTP/GDP-binding sites were predicted by flexible molecular docking approach which reveals that GTP/GDP binds to MtbEF-Tu mainly through hydrogen bonds.Our work lays the essential basis for further structural and functional studies of MtbEF-Tu as well as MtbEF-Tu-related novel drug developments.