Metal Ions Binding to RecA Inteins from Mycobacterium tuberculosis

摘要

Zinc has been found in the crystal structure of inteins and zinc ion can inhibit intein splicing both in vitro and in vivo. The interactions between metal ions and three minimized recA inteins have been studied in this work. Isothermal titration calorimetry (ITC) results show that the zinc binding affinity to three inteins is in the order of ΔI-SM > ΔΔIhh-SM ~ΔΔIhh-CM, but much weaker than to EDTA. These data explain the reversible inhibition and the presence of zinc only in the crystal structure of ΔI-SM of recA intein. A positive correlation between binding constants and inhibition efficiency was observed on the titration of different metal ions. Single-site binding mode was detected in all interactions, except ΔΔIhh-CM which has two Zn sites. Zinc binding sites on ΔΔIhh-CM were analyzed by NMR and ITC titration on inteins with chemical modifications. Results indicate that the Cys1 and His73 are the second zinc binding sites in ΔΔIhh-CM. CD study shows the metal coordinations have negligible influence on protein structure. This work suggests that the mobility restriction of key residues from metal coordination is likely the key cause of metal inhibition on intein splicing.