Cody C W, Huang P C
Department of Biochemistry, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland 21205-2103.
Biochemistry. 1993 May 18;32(19):5127-31. doi: 10.1021/bi00070a022.
Mammalian metallothioneins (MTs) possess eight highly conserved lysine residues, two of which constitute the hinge between two metal binding domains. By site-directed mutagenesis and recombinant DNA techniques, we replaced the interdomain lysines in Chinese hamster ovary MT2 with all possible combinations of glutamic acid and/or glutamine. The resultant MTs were expressed and assayed for detoxification function in a transformed yeast system. Results showed that these mutant MTs, like the native protein, bound seven atoms of divalent metal per molecule and conferred cadmium resistance to a metal-sensitive yeast host. Replacement of one or both of the lysines in the interdomain region was inconsequential to the structure and function of MT, unless both substituted residues were uncharged. When both lysines were replaced by glutamine (K30,31Q), a reduction in the ability of MT to protect yeast transformants against otherwise toxic levels of cadmium was observed. This diminished metal detoxification capacity was due to a decrease in the steady-state level of MT. These results suggest that at least one charged amino acid must be present in the hinge for the proper expression of MT.
哺乳动物金属硫蛋白（MTs）有八个高度保守的赖氨酸残基，其中两个构成两个金属结合结构域之间的铰链区。通过定点诱变和重组DNA技术，我们用谷氨酸和/或谷氨酰胺的所有可能组合替换了中国仓鼠卵巢MT2中结构域间的赖氨酸。在转化酵母系统中表达所得的MTs并检测其解毒功能。结果表明，这些突变型MTs与天然蛋白一样，每个分子结合七个二价金属原子，并赋予对金属敏感的酵母宿主镉抗性。结构域间区域中一个或两个赖氨酸的替换对MT的结构和功能没有影响，除非两个被取代的残基都不带电荷。当两个赖氨酸都被谷氨酰胺取代（K30,31Q）时，观察到MT保护酵母转化体免受镉毒性水平影响的能力下降。这种金属解毒能力的降低是由于MT稳态水平的下降。这些结果表明，铰链区中必须至少存在一个带电荷的氨基酸才能使MT正确表达。
