Structure- Function Relations and Liposomes Binding Features of DnaA from Mycobacterium tuberculosis.

DnaA is the primary initiator protein for DNA replication in bacteria. Accumulation of DnaA in bacteria, in several binding states position on the OriC region of the DNA convenes the DNA- replication proteins that constitute the replisome. Structure-function relationships of DnaA from Mycobacterium tuberculosis (Mtb), a pathogenic bacterium and the main causative agent of tuberculosis, is poorly studied.

Based upon the crystal structure of DnaA from Aquifex aeolicus the DnaA can be broadly divided into four functional segments: (1) N-terminal that associates with regulatory proteins, (2) helical linker region (3) ATP-binding domain that is a AAA+ (ATPase Associated with diverse cellular Activities) region and (4) DNA binding region in the C-terminal. In addition to DNA binding features, previous studies have shown that bacterial DnaA binds cytoplasmic membrane. Although not fully explored, such specific binding to the acidic phospholipids leads to activation of DnaA.

Our study aims to shed light on the differences between the DnaA bound states in the presence of several ligands and co-factors. Specifically, we compared the oligomeric state of DnaA upon binding of Mg(II), ATP, ADP, ADP-AlF₃ and DNA binding sequence (DnaA-box) in the presence of Mg(II) using SAXS, TEM, Cryo-EM, DLS, and AFM. A different oligomeric state is observed upon different binding states of DnaA. In addition, we show that the binding of DnaA to phospholipids (DMPC and DMPC-DMPG) leads to substantial changes in the conformation of DnaA and breaks the oligomers into sub-structures, mainly to monomers as observed by CD, TEM and AFM.