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

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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.