Characterization of a gas binding sensor heme kinase from M tuberculosis

DODES TRAIAN MM., OLIVIERI FA, MARTÍ MM, WETZLER DE

Congreso; XLV ReuniónAnnual Sociedad Argentina de Biofísica- IX IberoAmerican Congress of Biophysics. III Latin American Federation of Biophysical Societies; 2016

The two-component system DosST/R controls the entry ofMycobacterium tuberculosis and other mycobacteria into a latent, dormant state.Two clinically relevant conditions of the dormant state areits increased resistance to the effect of antibiotics and the relief of thesymptoms associated with the acute phase of infection: leading to an early stopof the treatment in affected patients and the consequent increase in thereports of multi-drug resistant strains favored by incomplete treatmentregimes.Hypoxia and exposure to NO and CO are stimuli thatphysiologically induce this dormant states. DosS and DosT are histidin kinaseswhich can sense gases through an heme-binding GAF domain and elicit the biologicalresponse through autophosphorylation of a histidine residue and then transferof this phosphate to the response element DosR. Structurally, these 62 kDaproteins present a modular structure consisting of two consecutive GAF domains,a dimerization domain (which also exposes the phosphorilable histidine residue)and an ATP binding domain.Only the N terminal GAF domain (GAF-A) binds heme and actsas a hipoxia and NO and CO sensor. The role of the GAF-B domain remains unknownand no ligand has been reported.We cloned and expressed DosS and DosT in Escherichia coli,and we have been able to purify and generate deletion mutants of DosS. We performeda biophysical characterization and analyzed the binding of gases on full DosS. Theautophosphorylation activity of the Kinase Core (dimerization plus ATP bindingdomain) was also measured. Our objective is being able to characterize thebiophysics and biochemistry of this system to gain insight into the allostericregulation by binding of gases and transduction of the signal from the firstGAF domain to the kinase core.