| In recent years,the incidence of Mycobacterium avium complex(Mac)-lung disease has continued to grow globally,and Mac's natural resistance to many commonly used drugs has made the treatment of Mac-lung disease ineffective.Trans-translation is essential for the reproduction and survival of bacteria,which is one of the important targets for drug development.Lon proteases recognize and degrade SsrA-tagged abnormal proteins during trans-translation to prevent abnormal proteins from forming toxic aggregates,thereby ensuring normal cell growth.In addition,Lon protease can specifically recognize different degrons to selectively degrade misfolded proteins and short-lived regulatory proteins to control the quality of proteins and maintain cell homeostasis.At present,the mechanism about how Lon proteases specifically recognize and bind substrates is known little,and the binding sites and interaction mode of Lon protease to recognize substrates are still unclear:The N-terminal domain of Lon protein is related to the specific recognition and binding of the substrate.It can be divided into three subdomains,and the third subdomain is considered to be involved in the oligomerization of Lon protease.In this study,we focus on the first two subdomains of Mycobacterium avium complex Lon protease N-terminal domain,aiming to solve the crystal structure of this fragment and complete the chemical shifts assignments of backbone atoms to further study the interaction between Lon protein and SsrA tag by using NMR technique,and to provide a structural and dynamics basis for Lon protease N-terminal domain to recognize and bind the other substrates.In this paper,the crystal structure of MacLon-N192 was successfully solved by X-ray diffraction technique at a resolution of 2.4 A.The structure consists of two distinct subdomains,the first subdomain consists of six ?-strands and one a-helix,and the second subdomain consists of five a-helices,very similar to the N-terminal fragment of Escherichia coli Lon protease,even though their sequence identity is only 26%.The comparision and analysis of sequence and structure indicated that the diversities may contribute to the substrate selectivity by Lon proteases from different species.The analyses of sequence conservation and structural hydrophobicity revealed that the recognition and binding between Lon proteases and substrates may be related to hydrophobic interactions.We also completed more than 88%chemical shift assignments of 1H-15N HSQC of MacLon-N192,and carried out R1,R2,and H-15N NOEs experiments based on the assignments.The results showed that the residues which are mainly located in the loops of crystal structure(I71-T76,S93-A98 and A109-T112)exhibited distinct flexible features,being consistent with the crystal structure of the MacLon-N192.In addition.The relaxation values revealed two relatively rigid loops L?1?2 and L?3?a in which the L?1?2 is stabilized by the interactions with two subdomains and the L?3?4 is stabilized by H-bond,salt-bridge,hydrophobic interactions and specific prolines.On the other hand,we confirmed that MacLon-N192 had no interaction with the SsrA tag by NMR titration,fluorescence polarization,isothermal titration calorimetry,and co-crystallization with FITC-SsrA.There are two possible reasons for it.First,the full-length Lon protease functions as a hexamer,while the MacLon-N192 protein exists as a monomer;and second,the sites that Lon proteases bind the SsrA tag may not be in the N-terminal domain.Our research on the interaction between MacLon-N192 and SsrA tag provides a reference for the study of the interaction between Lon protease and SsrA tag.More importantly,our research on the structure,backbone assignments,and dynamics analyses of MacLon-N 192 will provide a structural and dynamic basis for the specific recognition and binding of Lon protease N-terminal domain to other degrons or substrates. |
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