Javelle Arnaud, Lupo Domenico, Zheng Lei, Li Xiao-Dan, Winkler Fritz K, Merrick Mike
Department of Molecular Microbiology, John Innes Centre, Colney Lane, Norwich, Norfolk, NR4 7UH, United Kingdom.
J Biol Chem. 2006 Dec 22;281(51):39492-8. doi: 10.1074/jbc.M608325200. Epub 2006 Oct 12.
Amt proteins constitute a class of ubiquitous integral membrane proteins that mediate movement of ammonium across cell membranes. They are homotrimers, in which each subunit contains a narrow pore through which substrate transport occurs. Two conserved histidine residues in the pore have been proposed to be necessary for ammonia conductance. By analyzing 14 engineered polar and non-polar variants of these histidines, in Escherichia coli AmtB, we show that both histidines are absolutely required for optimum substrate conductance. Crystal structures of variants confirm that substitution of the histidine residues does not affect AmtB structure. In a subgroup of Amt proteins, found only in fungi, one of the histidines is replaced by glutamate. The equivalent substitution in E. coli AmtB is partially active, and the structure of this variant suggests that the glutamate side chain can make similar interactions to those made by histidine.
Amt蛋白构成了一类普遍存在的整合膜蛋白，介导铵离子跨细胞膜的转运。它们是同源三聚体，其中每个亚基都含有一个狭窄的孔道，底物通过该孔道进行转运。孔道中的两个保守组氨酸残基被认为是氨传导所必需的。通过分析大肠杆菌AmtB中这些组氨酸的14种工程化极性和非极性变体，我们发现这两个组氨酸对于最佳底物传导都是绝对必需的。变体的晶体结构证实，组氨酸残基的取代不会影响AmtB的结构。在仅在真菌中发现的Amt蛋白亚组中，其中一个组氨酸被谷氨酸取代。大肠杆菌AmtB中的等效取代具有部分活性，并且该变体的结构表明谷氨酸侧链可以与组氨酸产生类似的相互作用。
