Heme-based sensor kinases in Methanosarcina acetivorans as a paradigm for archaeal signal transduction

甲基硫化物甲基转移酶相关传感器MsmS(MA4561)来自产甲烷古细菌，是一个由交替的PAS和GAF结构域连接到C末端组氨酸激酶样H_ATPase输出域的多结构域传感器蛋白。MSMS在其第二个GAF结构域中包含一个共价结合的血红素辅因子。这种血红素具有氧化还原活性，能够连接几个外部配体，包括一氧化碳和二甲基硫化物。当血红素辅因子被氧化时，H_ATPase结构域的自磷酸化活性被诱导，表明MSMS是一种感受性激酶。借助于MSM的染色体敲除突变体，我们可以进一步证明MSMS参与了三个编码皮质醇/甲基转移酶同工酶的基因之一的交叉调节。这些基因的转录被证明是由MSR家族的上游编码调节子激活的。其中一个调节子被编码在MSM的下游，与之形成一个双顺反子操纵子，表明它们形成了一个双组分系统。在这项资助计划中，我们希望研究MSMS和同源激酶MA0863及其在皮质醇/甲基转移酶同工酶调节中的作用。一条研究路线将描述来自古生代的这一典型的血红素传感器激酶中的血红素的配位结构，具体而言，将检查共价键的作用。初步结果表明，MA0863在其第二个GAF结构域中也有一个血红素结合。将调查氧化还原性质和轴向配位是否与MSM中发现的血红素不同。因此，将采用紫外可见光谱、共振拉曼光谱和磁性圆二色光谱相结合的方法。氧化还原电位将通过氧化还原滴定实验来确定。虽然这两个传感器的氨基酸序列有很高的同源性，但MA0863的特殊之处在于它在其第二个PAS结构域编码一个琥珀密码子，该密码子在乙酰食单胞菌中被翻译成氨基酸吡咯赖氨酸。吡咯赖氨酸是否作为一种常规氨基酸残基或具有特殊功能是本提案的另一个目标。从感受器激酶的信号转导途径和皮质醇/甲基转移酶同工酶可能的交叉调节将被研究。除了编码皮质醇/甲基转移酶同工酶的靶基因的转录调控外，我们还希望探讨MSR调节因子在信号转导过程中的作用。这将通过传感器蛋白激酶和调节蛋白之间的蛋白质-蛋白质相互作用研究和使用凝胶迁移率变化分析的DNA-调节蛋白相互作用研究来完成。总体而言，我们希望对古生菌信号转导的一般理解有所贡献，尤其是对产甲烷生理学的理解。

The methyl sulfide methyltransferase associated Sensor MsmS (MA4561) from the methanogenic archaeon Methanosarcina acetivorans is a multidomain sensor protein consisting of alternating PAS and GAF domains joint to a C-terminal histidine kinase-like H_ATPase output domain. MsmS contains a covalently bound heme-cofactor in its second GAF domain. This heme is redox active and has the ability to ligate several external ligands including carbon monoxide and dimethyl sulfide. Upon oxidation of the heme cofactor autophosphorylation activity of the H_ATPase domain is induced suggesting that MsmS is a sensor kinase. With the help of a chromosomal knock out mutant of msmS we could further show that MsmS is involved in the cross-regulation of one of three gene encoding corrinoid/methyltransferase isozymes. The transcription of these genes was shown to be activated by upstream encoded regulators of the Msr family. One of these regulators is encoded downstream of msmS with which it forms a bicistronic operon suggesting that they form a two component system.Within this grant proposal we wish to investigate MsmS and the homologous kinase MA0863 and their role in regulating the corrinoid/methyltransferase isozymes. One line of investigation will characterize the coordination structure of the heme within this prototypical heme sensor kinase from Archaea and specifically the role of the covalent bond will be examined. Preliminary results indicated that MA0863 has also a heme bound in its second GAF domain. It will be investigated whether the redox properties and the axial coordination is different from the heme found in Msms. Therefore, a combination of UV-vis, resonance Raman und magnetic circular dichroism spectroscopy will be employed. Redox potentials will be determined using redox titration experiments. Although both sensors share high amino acid sequence homology, MA0863 is special in that it encodes an amber codon in its second PAS domain which is translated to the amino acid pyrrolysine in M. acetivorans. Whether pyrrolysine serves as a regular amino acid residue or holds a special function is another objective of this proposal. The signal transduction pathways from the sensor kinases and possible cross regulation of the corrinoid/methyltransferase isozymes will be investigated. Besides the transcriptional regulation of the target genes encoding the corrinoid/methyltransferase isozymes, we also wish to explore the function of the Msr regulators in the signal transduction process. This will be done with the help of protein-protein interaction studies between the sensor kinases and the regulator proteins and DNA-regulator interaction studies using gel mobility shift assays. Overall, we wish to contribute to the understanding of archaeal signal transduction in general but on methanogen physiology in particular.