Membrane Occupancy-Dependent Rejuvenation of DnaA Is Associated with Its Conformationally Driven Oligomerization

Abstract

DnaA, the initiator of chromosome replication in all known eubacteria species, is activated once per cell division cycle. Its overall activity cycle is driven by nucleotide exchange and ATP hydrolysis. Acidic phospholipids in a fluid membrane were shown to promote the rejuvenating nucleotide exchange on DnaA. We have recently shown that the transition into an active form is strongly cooperative with respect to DnaA membrane occupancy. Only at low membrane occupancy DnaA reactivation is efficiently catalyzed by the acidic phospholipids. The present study is aimed at unraveling the molecular outcome of the occupancy dependent DnaA rejuvenation. The comparison with N-terminal truncated protein, tDnaA, the specific labeling of DnaA by the environmentally sensitive fluorophore 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS), the CD examination of its secondary structure as well as the cross-linking at the N-terminal of DnaA revealed that: (i) DnaA N-terminal is indispensable in the cooperative transformation between the high and low occupancy states (I and II, respectively), (ii) the transformation between these states is associated with a conformational change, presumably at the N-terminal domain and (iii) State II of the protein on the membrane corresponds to a trimeric or higher form of DnaA. It is suggested that the DnaA conformation attained at low surface density drives its oligomerization which is presumably a pre-requisit to its interaction with oriC.