Probing the nucleotide binding and phosphorylation by the histidine kinase of a novel three-protein two-component system from Mycobacterium tuberculosis

Abstract 

The two-component signal transduction system from Mycobacterium tuberculosis bears a unique three-protein system comprising of two putative histidine kinases (HK1 and HK2) and one response regulator TcrA. By sequence analysis, HK1 is found to be an adenosine 5′-triphosphate (ATP) binding protein, similar to the nucleotide-binding domain of homologous histidine kinases, and HK2 is a unique histidine containing phosphotransfer (HPt)-mono-domain protein. HK1 is expected to interact with and phosphorylate HK2. Here, we show that HK1 binds 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate monolithium trisodium salt and ATP with a 1:1 stoichiometric ratio. The ATPase activity of HK1 in the presence of HK2 was measured, and phosphorylation experiments suggested that HK1 acts as a functional kinase and phosphorylates HK2 by interacting with it. Further phosphorylation studies showed transfer of a phosphoryl group from HK2 to the response regulator TcrA. These results indicate a new mode of interaction for phosphotransfer between the two-component system proteins in bacteria.

Abbreviations: ATP, adenosine 5′-triphosphate, HK1, histidine kinase 1, protein encoded by Rv0600c from Mycobacterium tuberculosis, HK2, histidine kinase 2, protein coded by RV0601c from M. tuberculosis, HPt, histidine containing phosphotransfer, IPTG, isopropyl β-d-galactoside, NADH, β-nicotinamide adenine di-nucleotide, reduced form, Ni-NTA, nickel-nitriloacetic acid, RR, response regulator, TcrA, response regulator coded by Rv0602c from M. tuberculosis, TNP-ATP, 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate monolithium trisodium salt

Keywords: TNP-ATP, Fluorescence, ATP binding, ATPase assay, Phosphotransfer, Response regulator