Studies On The Catalytic Mechanism Of Mycobacterium Tuberculosis Aminoacyl-tRNA Synthetase

Tuberculosis(TB)is a serious infectious disease caused by the human pathogen Mycobacterium tuberculosis,which can be transmitted from person to person through the air.Tuberculosis is one of the top 10 causes of death worldwide.According to the report from the World Health Organization,in 2016,10.4 million people have tuberculosis worldwide,1.8 million people died from the disease(400,000 of whom have HIV).Multidrug-resistant tuberculosis(MDR-TB)is a form of TB caused by the bacteria that do not respond to isoniazid and rifampicin,the two most powerful anti-TB drugs.In 2016,WHO estimates that there were 600,000 new cases worldwide.These new cases are an unprecedented challenge for the prevention and treatment of tuberculosis.The development of new antibiotics are urgently needed for the treatment of TB.An aminoacyl-tRNA synthetase(aaRS),an intracellular housekeeping protein,involved in the translation of genetic information into effector molecules(proteins),is an enzyme that attaches the appropriate amino acid onto its tRNA,ensures faithful translation of genetic information.An aaRS are divided into two classes,class I and class ? aaRS,based on structural and biochemical data:sequence similarity,the fold of catalytic domain,the way of interaction with tRNA.Methionyl-tRNA synthetase(MetRS)plays a critical role in protein synthesis.It is unique among essential aaRS because MetRS not only charges Met to tRNA for elongation of translation but also charges initiator tRNA to initialize translation.Moreover,MetRSs isolated from a variety of sources(species)show structural diversity.MetRS is considered as one of the most promising drug targets in combating bacterial pathogens,including Mycobacterium tuberculosis.There is no new report on the study of Mycobacterium tuberculosis Methionyl-tRNA synthetase(MtMetRS)since the K.value for aminoacylation reaction was reported at the end of the last century.This is related to the difficulties in the preparation of recombinant proteins.Due to the strong specificity of the MetRS inhibitor,the coverage of the antibacterial spectrum is narrow.Designing inhibitors based on structural requires accurate MtMetRS molecular model.However,the statistics to resolved MetRS structures show that only one of the eight species MetRS I enzymes has obtained a unliganded structure,and three of the four species MetRS II enzymes have obtained a unliganded structure.The statistics and literatures showed that unliganded MetRS I enzymes are difficult to obtain diffraction-quality crystals.MtMetRS belongs to the MetRS I.Therefore,the structural studies of MtMetRS should solve the problems of recombinant protein preparation and protein crystallization.We constructed different recombinant plasmids for recombinant protein expression screening,finally,solved the problem of preparation of MtMetRS recombinant protein.The kinetic of MtMetRS was first reported.Through crystal screening and optimization of crystallization conditions,the structure of unliganded MtMetRS and the crystal structure of the complex(MtMetRS:Met-AMP)binding to the intermediate Met-AMP were resolved.Structural analysis revealed that the MtMetRS:Met-AMP crystal structure has a similar Ca frame with other resolved MetRS.The analysis of resolved MetRS structures shows the catalytic domain of unliganded structures and their complexes have similar conformations.The catalytic domains are closed,forming a complete hydrophobic region.The substrate binding pocket remains in catalytic state at all times.However,the crystal structure of unliganded MtMetRS shows that its catalytic domain adopts a different conformation that of the complex structure.The catalytic domain of the unliganded MtMetRS is in an open state and does not form a closed hydrophobic region.Nucleotide binding loop(NBL)composed of hydrophobic amino acids is exposed to the water solvent and is in a collapsed state under hydrophobic effect.The structural comparison of the unliganded MtMetRS and the complex MtMetRS:Met-AMP shows that the substrate binding induces a conformational change in its catalytic domain.The changes occur at three sites:1.Nucleotide binding loop moves from the bottom of the catalytic pocket to the mouth;2.Substrate binding induces down-pressure of the CP(connective peptide)domain and stabilizes a2 helix;3.Structural rearrangement of ?-sheet of the Rossmann fold.These conformational changes make the catalytic domain of MtMetRS to form a hydrophobic region.Comparing MetRSs of different species,it was found that the resolved structures use the lock and key model to catalyze chemical reactions,in contrast with the resolved structures,the M.tuberculosis MetRS uses the Induced Fit model to catalyze chemical reactions.The results of biochemical experiments showed that the affinity and catalytic efficiency of MtMetRS to substrate were lower than that of E.coli MetRS under the same reaction conditions.The mutations showed that the stability of a2 helix had a great influence on the activity of MtMetRS.It is speculated that the stability of a2 affects the integrity of the hydrophobic core,thereby,affects the catalytic activity of the enzyme.The stability of a2 is related to the conformational change of the CP domain,which may be the reason why the MtMetRS uses the Induced Fit model to catalyze chemical reactions.The analysis of the distribution of conserved amino acids shows that the substrate binding region and tRNA antisense codon binding region consists of highly conserved amino acid.The amino acid that make up the tRNA amino acid acceptor arm binding region show less conservation.This provides clues for the design of specific inhibitors.In addition,we also found two the specific hydrophobic pockets existing between the nucleotide binding loop and ?10 or between ?2 and ?3 in the unliganded MtMetRS structure that are related to the conformational changes of MtMetRS.Occupation of these two hydrophobic pockets can inhibit substrate-induced MtMetRS conformational changes.These two hydrophobic pockets are good targets for inhibitor design.Based on results of the experimental and structural analysis,we propose that the MtMetRS employs a catalytic mechanism model different from the known MetRS:MtMetRS employs an induced-fit mechanism to catalyze substrates.By comparing with human cytoplasmic MetRS,we pointed out the hydrophobic binding pocket unique to MtMetRS that related to the catalytic activity of the enzyme.We provide accurate models and scientific evidence for structure-based rational design of inhibitors against Mycobacterium tuberculosis.