Characterization of the extracellular domain from Mycobacterium tuberculosis serine/threonine protein kinase PknF and functional implications

• Grant number: 21/10565-1
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: February 01, 2022
• End date: January 31, 2024
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Andrea Balan Fernandes
• Grantee: Natalia Von Staa Mansur
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Mycobacterium tuberculosis has eleven serine/threonine kinases, of which PknF has been associated with the regulation of the ABC-type transporter (ATP-Binding Cassette) Rv1747, whose function is still unknown. PknF has two domains connected by a transmembrane helix, the extracellular domain (sensor, ePknF) and the intracellular domain (catalytic, cPknF). Unlike other canonical ABC transporters that consist of two transmembrane permeases and two cytoplasmic ATPases, Rv1747 contains two additional Fork-Head Associated-like domains (FHA1 and FHA2) connected by an unstructured 150-amino acid region (linker). Different studies have shown that cPknF activates the transporter by phosphorylating two threonine residues (T152 and T210) present in the linker, in addition to being associated with reduced glucose uptake and mycolic acid synthesis by the bacillus, both characteristic responses of the infection process and latency in the macrophage. In the group's work, we solved the structure of the catalytic domain and showed that the phosphorylation of threonine residues in the linker between the FHA domains, promoted by cPknF, promotes their oligomerization, significant structural changes, and consequent activation of the transporter. We also found that cPknF activity is related to the dimeric state, which in other works with PknF-like kinases, is related to the sensory role of the extracellular domain. Considering the intriguing role that PknF activity has on bacilli infection and the lack of data on the mechanisms of activation of the transporter, this project aims to structurally characterize the ePknF domain and understand the possible cellular stimuli that lead to dimerization and activation of the kinase. As a methodology, bioinformatics tools, domain expression, and purification will be used for biophysical studies such as circular dichroism, dynamic light scattering, and SAXS, in addition to domain crystallization and resolution of the three-dimensional structure. Additionally, to identify possible ePknF partner molecules and changes in their structure, pull-down domain assays with macrophage cell extracts, calorimetry assays in the presence of ligands, and molecular docking assays and molecular dynamics of PknF in the presence and absence of possible ligands.(AU)