Elucidating the cis/trans autophos-phorylation mechanism of the DosS sensor histidine kinase from Mycobacterium tuberculosis

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

La Plata

Congreso; XLVII Reunión Anual de la Sociedad Argentina de Biofísica; 2018

Elucidating the cis/trans autophosphorylation mechanism of the DosS sensor histidine kinase from Mycobacterium tuberculosisF.Olivieri, M. Dodes Taria, M.Marti, D.E.WetzlerTwo-component systems (TCSs), comprising histidine kinases (HKs) and response regulators, empower bacteria to sense and adapt to diverse environmental stimuli. The TCS DosS-DosT/DosR from Mycobacterium tuberculosis controls the entry of the bacillus into a latent, dormant state, that renders antibiotics inefficient while reducing clinical manifestations of the disease. DosS and DosT are heme-histidine kinases which can be activated by hypoxia or by the presence of NO or CO. When active, these HKs undergo autophosphorylation in a conserved histidine residue and then transfer the phosphate to an aspartic residue in the DosR regulator, which induces the expression of the dormancy regulon.As most TCSs, DosS and DosT consist of a sensor domain and a kinase core (KC) with an ATP binding domain (ABD) and a dimerization histidine phosphate accepting domain (DHp). Structurally, dimeric HKs can autophosphorylate in a cis (intramonomer) or trans (intermonomer) 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.As the KC tertiary structures of DosS and DosT have not been solved yet, we elucidated the cis/trans mechanism performing a biochemical approach. For this purpose, we engineered DosS mutants without sensor domains, which are known to be constitutively active. One of these mutants also had two point-mutations that inhibit its ability to bind ATP, but still allows it to act as phosphate acceptor and another mutant lacks the phosphorylatable histidine but can bind ATP and act as a phosphate donor.Employing these constructs we elucidated that DosS undergoes autophosphorylation by a trans mechanism. Considering this result together with the available structural information for DosS and DosT and other HKs, we have constructed a homology model that will allow us to study the phosphorylation and activation mechanisms through classical and quantum molecular dynamics