Iwama Tomonori, Ito Yasuaki, Aoki Hisaaki, Sakamoto Hiroshi, Yamagata Shuzo, Kawai Keiichi, Kawagishi Ikuro
Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu 501-1193, Japan.
J Biol Chem. 2006 Jun 30;281(26):17727-35. doi: 10.1074/jbc.M601038200. Epub 2006 Apr 24.
The chemoreceptor Tcp of Salmonella enterica serovar Typhimurium can sense citrate and a metal-citrate complex as distinct attractants. In this study, we tried to investigate the molecular mechanism of this discrimination. That citrate binds directly to Tcp was verified by the site-specific thiol modification assays using membrane fractions prepared from Escherichia coli cells expressing the mutant Tcp receptors in which single Cys residues were introduced at positions in the putative ligand-binding pocket. To determine the region responsible for the ligand discrimination, we screened for mutations defective in taxis to magnesium in the presence of citrate. All of the isolated mutants from random mutagenesis with hydroxylamine were defective in both citrate and metal-citrate sensing, and the mutated residues are located in or near the alpha1-alpha2 and alpha3-alpha4 loops within the periplasmic domain. Further analyses with site-directed replacements around these regions demonstrated that the residue Asn(67), which is presumed to lie at the subunit interface of the Tcp homodimer, plays a critical role in the recognition of the metal-citrate complex but not that of citrate. Various amino acids at this position differentially affect the citrate and metal-citrate sensing abilities. Thus, for the first time, the abilities to sense the two attractants were genetically dissected. Based on the results obtained in this study, we propose models in which the discrimination of the metal-citrate complex from citrate involves cooperative interaction at Asn(67) and allosteric switching.
肠炎沙门氏菌鼠伤寒血清型的化学感受器Tcp能够将柠檬酸盐和金属 - 柠檬酸盐复合物感知为不同的引诱剂。在本研究中，我们试图探究这种区分的分子机制。通过使用从表达突变型Tcp受体的大肠杆菌细胞制备的膜组分进行位点特异性硫醇修饰试验，证实了柠檬酸盐直接与Tcp结合，其中在假定的配体结合口袋中的位置引入了单个半胱氨酸残基。为了确定负责配体区分的区域，我们在柠檬酸盐存在下筛选了对镁趋化性有缺陷的突变体。所有用羟胺进行随机诱变分离出的突变体在柠檬酸盐和金属 - 柠檬酸盐感知方面均有缺陷，并且突变残基位于周质结构域内的α1 - α2和α3 - α4环内或其附近。围绕这些区域进行定点置换的进一步分析表明，推测位于Tcp同型二聚体亚基界面的Asn(67)残基在金属 - 柠檬酸盐复合物的识别中起关键作用，而对柠檬酸盐的识别不起关键作用。该位置的各种氨基酸对柠檬酸盐和金属 - 柠檬酸盐的感知能力有不同影响。因此，首次从基因角度剖析了感知这两种引诱剂的能力。基于本研究获得的结果，我们提出了模型，其中从柠檬酸盐中区分出金属 - 柠檬酸盐复合物涉及Asn(67)处的协同相互作用和变构转换。
