Exploring the Cis/Trans Autophosphorylation Mechanism of a Sensor Histidine Kinase From Mycobacterium tuberculosis

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

Buenos Aires

Congreso; II Reunión Conjunta de Sociedades de Biociencias; 2017

One of the mechanisms that contributes to persistent infection byMycobacterium tuberculosis is the bacillus capacity to enter a latent,dormant state, that renders antibiotics inefficient while reducing clinical manifestations of the disease. The DosS-DosT/DosR two-component sensor system consists of the DNA binding element DosR that induces expression of the ~48 gene dormancy regulon and the signaling heme histidine kinases (HK) DosS and DosT. These signaling HKs can be activated by hypoxia, presence of NO, CO andascorbic acid. Following activation, HK autophosphorylate by transferring a phosphate moiety from an ATP molecule to a conserved histidine residue; this phosphate is then transferred to an aspartic acid residue in DosR. DosS is a HK consisting of an heme binding domain (GAF1), a GAF2 domain with unknown function and a dimerization-phosphorylation (DhP) domain. Structurally, dimeric HKscan autophosphorylate in a cis or trans intradimer mechanism. It has been posited that the key determinant is the loop that connects two alfa-helices at the base of the DhP domain four-helix bundle. In this case, the DosS DhP tertiary structure has not been solved yet. To explore this mechanism in DosS, we engineered mutants removing the sensor domains leaving just the DhP domain to ensure constitutive activity of the enzyme. We also engineered an impaired ATP-binding mutant to act as a possible phosphate acceptor but unable to transfer phosphate to a histidine residue. We verified structural integrity and tertiary and quaternary structure of our constructs by circular dichroism, size exclusion chromatography and dynamic light scattering. Autophosphorylation activity was verified by incorporation of radioactive phosphorus from ATP-γ-32P. Usingthese protein constructs and the information regarding their activity and quaternary structure we will present biophysical/biochemical evidence that will contribute to elucidate the mechanism of DosS autophosphorylation