title: Metal ions binding to recA inteins from Mycobacterium tuberculosis.
abstract: Zinc has been found in the crystal structures of inteins and the 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 DeltaI-SM > DeltaDeltaI(hh)-SM approximately DeltaDeltaI(hh)-CM, but is much weaker than to EDTA. These data explain the reversible inhibition and the presence of zinc only in the crystal structure of DeltaI-SM of recA intein. A positive correlation between binding constants and inhibition efficiency was observed upon the titration of different metal ions. Single-site binding modes were detected in all interactions, except DeltaDeltaI(hh)-CM which has two Zn sites. Zinc binding sites on DeltaDeltaI(hh)-CM were analyzed by NMR spectroscopy and ITC titration on inteins with chemical modifications. Results indicate that the Cys1 and His73 are the second zinc binding sites in DeltaDeltaI(hh)-CM. CD studies show 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 of intein splicing.
authors.affiliation: Department of Chemistry, University of Science and Technology of China, Hefei, Anhui, China. liuyz@ustc.edu.cn
authors.forename: Liyun
authors.initials: L
authors.lastname: Zhang
authors.forename: Yuchuan
authors.initials: Y
authors.lastname: Zheng
authors.forename: Zhaoyong
authors.initials: Z
authors.lastname: Xi
authors.forename: Zhaofeng
authors.initials: Z
authors.lastname: Luo
authors.forename: Xiaolong
authors.initials: X
authors.lastname: Xu
authors.forename: Chunyu
authors.initials: C
authors.lastname: Wang
authors.forename: Yangzhong
authors.initials: Y
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002104:Cadmium / Q000737:chemistry / Q000378:metabolism; D003035:Cobalt / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D047668:Inteins / Q000235:genetics*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D019216:Metals, Heavy / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009532:Nickel / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011938:Rec A Recombinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D019216:Metals, Heavy; D002104:Cadmium; D003035:Cobalt; D008345:Manganese; D009532:Nickel; D011938:Rec A Recombinases; D015032:Zinc