NMR Based Structure,Function And Dynamics Analysis Of Ribosomal Protein S1 From Mycobacterium Tuberculosis

Global tuberculosis report for 2016 noted that tuberculosis had hold the highest mortality than HIV/AIDS,remained one of the top 10 causes of death worldwide in 2015,while Mycobacterium tuberculosis(Mtb)is a pervasive human pathogen that causes tuberculosis(TB).Therefore,the identification of new targets in Mtb that might be inhibited to effectively kill the existing strains is now a global pursuit.Pyrazinamide(PZA),the first-line anti-tuberculosis drug,has been used in clinic for more than 60 years,with its prominent feature that can shorten the treatment period of TB from 9-12 months to 6 months.Recently,it found that pyrazinoic acid(POA),the active form of PZA,binds to the C-terminus of the ribosomal protein(MtRpsACTD)and inhibits trans-translation,which is essential for cell survival under stressful condition.To explore the nature of the MtRpsA-POA interaction and why POA exhibits an exquisite specificity to MtRpsA,the crystal structures of the MtRpsACTD alone and in complex with POA have been obtained.The results show that pyrazinoic acid can target the fourth S1 domian of MtRpsA to block the interaction of MtRpsA-tmRNA.However,it remains elusive how POA affects the dynamics of MtRpsA and how MtRpsA interacts with tmRNA,the molecular mechanism that is essential for understanding the MtRpsA function in trans-translation.In this study,we attempted to investigate the interactions of MtRpsA with either POA or tmRNA respectively by using multi-dimensional heteronuclear NMR spectroscopy to obtain the the molecular mechanism of POA involved in the interaction between MtRpsA and tmRNA in trans-translation and provide theoretical basis for the designment of new anti-TB drugs.Firstly,we got the full-length MtRpsA protein and explored its physicochemical properties by using molecular biology method and biochemical technique.We constructed the recombinants of MtRpsA and optimized persistently expression and purification conditions.Finally,we obtained a stable and homogeneous MtRpsA sample by one-step affinity purification following with gel chromatography.Furthermore,the physicochemical properties of proteins were preliminarily studied by using dynamic light scattering,circular chromatography and NMR technology.This work has laid the foundation for the further study of MtRpsA in trans-translation process.Then,we focused on the interaction between MtRpsACTD and POA,and how POA affects the dynamics of MtRpsACTD by using NMR HSQC titration.The results showed that the interaction between MtRpsACTD and POA is moderate.With the titrated POA being increased,the chemical shifting of the amino acids which involve the binding sites of POA-MtRpsACTD were perturbed obviously.At the same time,the chemical environments of a few unbinded residues were also changed due to the structural allosteric effect.The dynamic analysis revealed that POA did not change largely the overall rigidity structure property of the MtRpsACTD protein,but influenced its interior movement.Moreover,we analyzed the interaction of the C-terminal domain of MtRpsA with three RNA fragments poly(A),sMLD,and pre-sMLD.NMR titration analysis revealed that the RNA binding sites on MtRpsACTD are mainly located in the β2,β3,and P5-strands and the adjacent L3 loop of the S1 domain.Fluorescence experiments determined the MtRpsACTD binding to RNAs are in the micromolar affinity range.Sequence analysis also revealed conserved residues in the mapped RNA binding regions.Docking model of MtRpsACTD-pre-sMLD showed poly(A)-like sequences and sMLD of MLD together mediated the interaction of tmRNA and protein,with charge and hydrogen bond interaction as the main interaction forces.All in all,results showed that POA competed with tmRNA to interact with MtRpsACTD,with which both had same binding affinities ranging in μM and interaction binding sites.The findings not only provided the structural basis to explain how POA competed with tmRNA to interact with MtRpsACTD which blocks the trans-translation,but also provided the experimental data for the development of new anti-TB drugs and other new antibiotics.