Exploring the communication between the heme-binding GAF sensor and the kinase core in the histidine kinase DosS from Mycobacterium tuberculosis

MACERI, MICOL; WETZLER, DIANA E.; JELIC, EDUARDO; DODES TRAIAN, MARTÍN M.; OLIVIERI, FEDERICO A.; MARTÍ, MARCELO A.

Congreso; L Reunión Anual de la Sociedad Argentina de Biofísica; 2022

Sociedad Argentina de Biofísica

Two-component systems (TCSs) are the main systems used by bacteria to detect, respond and adapt to a large variety of environmental and intracellular signals. Prototypical TCSs consist of two proteins, a sensor histidine kinase (SHK) and an effector response regulator (RR). Depending on the stimulus, the SHK autophosphorylates at a conserved histidine residue. Subsequently, the phosphoryl group is transferred to a conserved aspartate residue of the RR. Interestingly, in several systems, the SHK itself acts as a phosphatase of their cognate phosphorylated RR. SHKs are generally homodimeric proteins, comprising an N-terminal sensor domain and a C- terminal more conserved kinase core (KC). Connecting the sensor domain and the KC there may be a HAMP, GAF or PAS domains, a tandem of this domains or small helices like the Jα helices. 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 SHKs undergo autophosphorylation and then transfer the phosphate to their RR DosR, which induce the expression of the dormancy regulon. DosS and DosT possess a heme-binding GAF sensor domain (GAFA), a secondary GAF domain of unknown function (GAFB) and the KC. Although this system has been extensively studied, many questions still remain open. Employing an in vitro biophysical and biochemical approach, we will focus particularly on the communication between the sensor domain (GAFA) and the KC and on the unknown function of GAFB. Based on this objective, we designed, cloned, purified and characterized different DosS constructs that will allow us to address the proposed approach