Metal ions binding to recA inteins from Mycobacterium tuberculosis.
Metal ions binding to recA inteins from Mycobacterium tuberculosis.
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DOI：
10.1039/b903144h
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发表时间：
2009-06
影响因子：
--
通讯作者：
Liu Y
中科院分区：
文献类型：
--
作者：
Zhang L;Zheng Y;Xi Z;Luo Z;Xu X;Wang C;Liu Y
锌离子存在于内含肽的晶体结构中，在体内外均能抑制内含肽的剪接。本文研究了金属离子与三种最小化recA内含肽的相互作用。等温滴定量热法（ITC）结果表明，三种内含肽对锌的结合亲和力顺序为ΔI-SM > ΔΔIhh-SM ~ΔΔIhh-CM，但远弱于EDTA。这些数据解释了可逆抑制和锌仅存在于recA内含肽的ΔI-SM晶体结构中。不同金属离子的滴定结果表明，结合常数与缓蚀效率呈正相关。除ΔΔIhh-CM有两个Zn位点外，其余均为单位点结合。通过NMR和ITC滴定分析了ΔΔIhh-CM上的锌结合位点。结果表明，Cys 1和His 73是ΔΔIhh-CM的第二个锌结合位点。圆二色谱研究表明金属配位对蛋白质结构的影响很小。这项工作表明，金属配位的关键残基的流动性限制可能是金属抑制内含肽剪接的关键原因。
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.
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