EXPLORING THE CIS/TRANS AUTOPHOSPHORYLATION MECHANISM OF A SENSOR HISTIDINE KINASE

MARCLEO MARTÍ, MARCELO A.; OLIVIERI, FEDERICO A.; DODES TRAIAN MARTIN M.; WETZLER, DIANA E.

Buenos Aires

Congreso; REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS; 2017

SOCIEDADES DE BIOCIENCIAS ARGENTINAS

One of the mechanisms thatcontributes to persistent infection by Mycobacteriumtuberculosis isthe bacillus capacity to enter a latent, dormant state, that rendersantibiotics inefficient while reducing clinical manifestations of the disease.The DosS-DosT/DosR two-component sensor system consists of the DNA bindingelement DosR that induces expression of the ~48 gene dormancy regulon and thesignaling heme histidine kinases (HK) DosS and DosT. These signaling HKs can beactivated by hypoxia, presence of NO, CO and ascorbic acid. Followingactivation, HK autophosphorylate by transferring a phosphate moiety from an ATPmolecule to a conserved histidine residue; this phosphate is then transferredto an aspartic acid residue in DosR. DosS is a HK consisting of an heme bindingdomain (GAF1), a GAF2 domain with unknown function and a dimerization-phosphorylation (DhP) domain. Structurally, dimeric HKs can autophosphorylatein a cis or trans intradimer mechanism. It has been posited that thekey determinant is the loop that connects two alfa-helices at the base of theDhP domain four-helix bundle.In this case, the DosS DhP tertiarystructure has not been solved yet. To explore this mechanism in DosS, weengineered mutants removing the sensor domains leaving just the DhP domain toensure constitutive activity of the enzyme. We also engineered an impairedATP-binding mutant to act as a possible phosphate acceptor but unable totransfer phosphate to a histidine residue. We verified structural integrity andtertiary and quaternary structure of our constructs by circular dichroism, sizeexclusion chromatography anddynamic light scattering. Autophosphorylationactivity was verified by incorporation of radioactive phosphorus fromradiactivo. Using these protein constructs and the information regarding theiractivity and quaternary structure we will present biophysical/biochemicalevidence that will contribute to elucidate the mechanism of DosS autophosphorylation.Keywords:Histidine Kinases, Mycobacteriumtuberculosis, autophosphorylation