Structural And Functional Study Of The N-terminal Fragment Of Lon Protease From Mycobacterium Tuberculosis Complex

Tuberculosis(TB)is a chronic infectious disease and has been one of the top 10 infectious diseases threatening human health.Anti-tuberculosis therapy is getting increasingly serious with the spread of drug-resistant TB bacteria,and finding new therapeutic targets and developing new anti-tuberculosis drugs are urgently required.In recent years,intracellular proteolysis has gradually become a research hotspot,and Lon protease is able to recognize and degrade abnormal proteins in cells.Lon protease regulates the reaction mechanism by participating in SOS and degrades the cell division inhibitor SulA protein generated in the reaction,so as to regulate the life processes such as cell division and transcription,and Lon protease may be treated as an important target for the treatment of tuberculosis.Lon protease is mainly divided into 3 domains,Nterminal domain,ATPase domain and protease domain.And its N-terminal domain can recognize and bind the substrate.Meanwhile Lon protease can recognize the protein carrying sul20C tag,but the molecular mechanism and structural information of its recognition and binding have not been clear yet.Therefore,the study of the structure and function of the N-terminal domain of Lon protease is conducive to revealing the key sites and mechanism of its recognition and binding substrate,and provides thoughts for the development of anti-tuberculosis drugs.In this paper,the structure and function of the Lon protease N-terminal domain fragment from Mycobacterium tuberculosis complex(MTBC)were studied,and the main progresses were as follows:(1)Recombinant plasmids containing Lon protease N-terminal domain fragments such as MTBCLon1-613,MTBCLon1-313,MTBCLon1-193 and MTBCLon1252,MTBCLon1-211 were successfully constructed using molecular cloning technology;and MTBCLon1-211 protein samples with high quality,good uniformity and high stability were obtained after a series of protein purification and physical and chemical characterization;(2)The MTBCLon1-211 protein samples were analyzed by NMR and CD,and the protein was well folded,mainly with a helix;2D1H-15N HSQC spectrum showed that the peak rate of target protein amide hydrogen(NH)was more than 90%(180/197),with the possibility of main chain identification and analysis of 3dimensional structure by NMR;(3)The crystal structure of MTBCLon1-211 protein was resolved by X-ray diffraction,with a resolution of 2.0 A,a space group of P121,and 2 protein molecules in an asymmetric unit with 180°rotational symmetry.In the refined model of MTBCLon1-211 protein,the Rwork/Rfree value was 0.202/0.239,which was successfully published in the protein database(PDB ID:6VBK).The crystal structure of MTBCLon1-211 protein was spherical.A single protein molecule consisted of 2 subdomains and a C-terminal truncated a helix,where the first subdomain consisted of an a helix and 6 β folds;and the second subdomain consisted of 4 α helices.This study provides experience for the protein structure of the Lon protease Nterminal domain and the interaction between the protein and the substrate,which in turn provides new research thoughts for drug development and inhibitor screening.