Study On Interaction Between Ribosome Maturation Factor M And Ribosomal Protein S19 From Mycobacterium Tuberculosis

Tuberculosis is a highly lethal chronic infectious disease caused by Mycobacterium tuberculosis.With the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis,traditional anti-tuberculosis drugs have been showing less efficiency.Therefore,finding new antibacterial drug targets has currently become a research hotspot in order to develop effective new anti-tuberculosis drugs.The biosynthesis of ribosomes is the basis of cellular forms of life.The absence of mature ribosomes will lead to cellular dysfunction or even a significant shortening of the life span,which shows the importance of the coordinated synthesis and assembly of ribosome components in prokaryotes.MtRimM(Mycobacterium tuberculosis Ribosome maturation factor M)is a member of the strain ATCC 25618/H37Rv,and can interact with the ribosomal protein S19 in the assembly process of 30S subunit as a ribosomal maturation factor protein.Therefore,MtRimM possesses the potency of being exploited as a novel antituberculosis drug target.However,the underlying molecular mechanism of the interaction between MtRimM and S19 remain unclear so far.Therefore,it is important to address molecular mechanisms of the interaction between MtRimM and S19 during the maturation of 30S ribosomal subunit in Mycobacterium tuberculosis.In this thesis,we studied the interaction between MtRimM and S19 by means of various techniques,including molecular biology tools,pull-down experiments,ITC experiments,BLI experiments,NMR experiments,and protein crystallization.First,we constructed optimal expression vectors of the target proteins(MtRimM,S19)and the N-terminal domain and C-terminal domain protein(MtRimMCTD,MtRimMNTD)of MtRimM using molecular cloning technique.Then,we established an efficient protocol by optimizing the conditions of protein expression,including expression temperature,inducer concentration,the salt concentration and imidazole concentration during CoNTA purification.Through the further purification with molecular chromatography(FPLC),we obtained the following proteins:MtRimM,highest yield 20 mg/mL,purity 95%;MtRimMCTD and MtRimMNTD,highest yield 25 mg/mL,purity 95%;S19,highest yield 30 mg/mL,purity 95%.Next,we characterized the proteins by DLS and CD spectroscopy and explored the stability of the proteins under different physicochemical conditions by means of 1D1H,2D 1H-15NHSQC NMR spectrum,which showed that the solution state of MtRimM,MtRimMCTD,and MtRimMNTD protein was uniform and they can be stably stored at room temperature(25℃)for at least one week and at-80℃for a long period.The protein S19 has a uniform solution state as well,and can be stored at 4℃ for 4 days.MtRimMCTD and S19 can also be lyophilized and redissolved into buffer,which does not affect their backbone structural characteristics.After these assays,we determined the existence of interaction between MtRimM and S19 by molecular sieve and pull down experiments,so was the interaction between MtRimMCTD and S19.We studied interaction by using the ITC technology on a thermodynamic level.The result shows a moderately strong level of interaction between MtRimM and S19 for the dissociate constant(Kd)was 10-7 M and a moderate level of interaction between MtRimMCTD and S19 for the Kd was 10-6 M.The BLI technology was used to study the interaction of them on a kinetic level and the results were basically consistent with those of ITC that Kd was 10-7 M for MtRimM and S19 and Kd was 10-6 M for MtRimMCTD and S19.We also verified the interaction between MtRimMCTD and S19 by NMR titration.Based on 1H-15N HSQC NMR spectrum,the chemical shift changes of 7 peaks(L12,V28,L47,A53,S57,S59,167)are statistically significant by calculating the chemical shift perturbation value of each peak and there are another 30 peaks that obviously broadened or even disappeared after titrating 15N-MtRimMCTD with S19 without artificial isotope label.Combined with the conservative amino acid position analysis achieved by multiple sequence alignment and analysis of the crystal structure of the complex of RimM and S19 in the Thermus thermophilus that amino acid sites within 5 ? of the interatomic distance,it is reasonable to assume that 13 sites(Y4、Y5、L9、D6、H30、T31、A33、G34、E35、L36、V48、V28、L47)werepotentially involved in the interaction between MtRimM and S19.Nevertheless,this conclusion is being verified by site-directed mutation.On the other hand,we determined that MtRimMNTD did not interact both with MtRimMCTD and with S19 with ITC and NMR titration experiments.Furthermore,we obtained MtRimMNTD crystals.However,the crystallization conditions needed to be optimized.Besides,we screened a variety of conditions for crystallizing MtRimM monomers and MtRimM-S19 composite proteins.So far these conditions did not work yet.We will continue to screen the crystallization conditions in near future.Our results lay the foundation for determination of the 3D structure of the MtRimM-S19 complex and clarification of the molecular mechanism underlying the protein-protein interaction.This work may be of benefit to further understanding of biological functions of MtRimM.