Tyrrell R, Verschueren K H, Dodson E J, Murshudov G N, Addy C, Wilkinson A J
Department of Chemistry, University of York, Heslington, UK.
Structure. 1997 Aug 15;5(8):1017-32. doi: 10.1016/s0969-2126(97)00254-2.
CysB is a tetrameric protein of identical subunits (M(r) = 36,000) which controls the expression of genes associated with the biosynthesis of cysteine in bacteria. CysB is both an activator and a repressor of transcription whose activity is responsive to the inducer N-acetylserine; thiosulphate and sulphide act as anti-inducers. CysB is a member of the LysR family of prokaryotic transcriptional regulatory proteins which share sequence similarities over approximately 280 residues including a putative helix-turn-helix DNA-binding motif at their N terminus. The aims of the present study were to explore further the complex molecular biology and curious ligand binding properties of CysB and to provide structural insights into the LysR family of proteins.
The crystal structure of a dimeric chymotryptic fragment of Klebsiella aerogenes CysB comprising residues 88-324, has been solved by multiple isomorphous replacement and multi-crystal averaging and refined against data extending to 1.8 A resolution. The protein comprises two alpha/beta domains (I and II) connected by two short segments of polypeptide. The two domains enclose a cavity lined by polar sidechains, including those of two residues whose mutation is associated with constitutive expression of the cysteine regulon. A sulphate anion and a number of well ordered water molecules have been modelled into discrete electron-density peaks within this cavity. In the dimer, strands beta B from domain I and strands beta G from domain II come together so that a pair of antiparallel symmetry-related 11-stranded twisted beta-pleated sheets is formed.
The overall structure of CysB(88-324) is strikingly similar to those of the periplasmic substrate-binding proteins. A similar fold has also been observed in the cofactor-binding domain of Lac repressor, implying a structural relationship between the Lac repressor and LysR families of proteins. In contrast to Lac repressor, in CysB the twofold axis of symmetry that relates the monomers in the dimer is perpendicular rather than parallel to the long axis of the cofactor-binding domain. This seems likely to place the DNA-binding domains at opposite extremes of the molecule possibly accounting for CysB's extended DNA footprints.
CysB是一种由相同亚基组成的四聚体蛋白（分子量为36,000），它控制着细菌中与半胱氨酸生物合成相关基因的表达。CysB既是转录激活因子又是转录抑制因子，其活性对诱导剂N - 乙酰丝氨酸有反应；硫代硫酸盐和硫化物作为抗诱导剂。CysB是原核生物转录调节蛋白LysR家族的成员，该家族在大约280个残基上具有序列相似性，包括其N端一个假定的螺旋 - 转角 - 螺旋DNA结合基序。本研究的目的是进一步探索CysB复杂的分子生物学和奇特的配体结合特性，并为LysR家族蛋白提供结构上的见解。
通过多同晶置换和多晶体平均法解析了产气克雷伯菌CysB包含88 - 324位残基的二聚体胰凝乳蛋白酶片段的晶体结构，并根据分辨率延伸至1.8 Å的数据进行了精修。该蛋白由两个α/β结构域（I和II）通过两段短的多肽链连接而成。这两个结构域包围着一个由极性侧链排列的腔，包括两个其突变与半胱氨酸调节子的组成型表达相关的残基。一个硫酸根阴离子和一些排列有序的水分子已被模拟到这个腔内离散的电子密度峰中。在二聚体中，结构域I的βB链和结构域II的βG链聚集在一起，从而形成一对反平行的对称相关的11股扭曲β折叠片层。
CysB(88 - 324)的整体结构与周质底物结合蛋白的结构惊人地相似。在乳糖阻遏蛋白的辅因子结合结构域中也观察到了类似的折叠，这意味着乳糖阻遏蛋白和LysR家族蛋白之间存在结构关系。与乳糖阻遏蛋白不同，在CysB中，二聚体中与单体相关的二重对称轴垂直于而不是平行于辅因子结合结构域的长轴。这似乎可能使DNA结合结构域位于分子的相反两端，这可能解释了CysB扩展的DNA足迹。
