Characterization Of A Gas Binding Sensor Heme Kinase from Mycobacterium tuberculosis.

MARTÍN MIGUEL DODES TRAIAN; FEDERICO OLIVIERI; MARCELO MARTÍ; DIANA WETZLER

San Miguel de Tucumán

Congreso; XLV Reunión Anual Sociedad Argentina de Biofísica; 2016

Sociedad Argentina de Biofísica

The two-component system DosST/R controls the entry of Mycobac-terium tuberculosis and other mycobacteria into a latent, dormant state.Two clinically relevant conditions of the dormant state are its increasedresistance to the effect of antibiotics and the relief of the symptoms as-sociated with the acute phase of infection: leading to an early stop of thetreatment in affected patients and the consequent increase in the reportsof multi-drug resistant strains favored by incomplete treatment regimes.Hypoxia and exposure to NO and CO are stimuli that physiologicallyinduce this dormant states. DosS and DosT are histidin kinases whichcan sense gases through an heme-binding GAF domain and elicit thebiological response through autophosphorylation of a histidine residueand then transfer of this phosphate to the response element DosR. Struc-turally, these 62 kDa proteins present a modular structure consisting oftwo consecutive GAF domains, a dimerization domain (which also ex-poses the phosphorilable histidine residue) and an ATP binding domain.Only the N terminal GAF domain (GAF-A) binds heme and acts as ahipoxia and NO and CO sensor. The role of the GAF-B domain remainsunknown and no ligand has been reported.We cloned and expressed DosS and DosT in Escherichia coli, and wehave been able to purify and generate deletion mutants of DosS. Weperformed a biophysical characterization and analyzed the binding ofgases on full DosS. The autophosphorylation activity of the Kinase Core(dimerization plus ATP binding domain) was also measured. Our ob-jective is being able to characterize the biophysics and biochemistry ofthis system to gain insight into the allosteric regulation by binding ofgases and transduction of the signal from the first GAF domain to thekinase core.Acknowledgments: CONICET , ANPCyT and UBACyT