The structure of a DnaA/HobA complex from Helicobacter pylori provides insight into regulation of DNA replication in bacteria

Abstract

Bacterial DNA replication requires DnaA, an AAA؉ ATPase that initiates replication at a specific chromosome region, oriC, and is regulated by species-specific regulators that directly bind DnaA. HobA is a DnaA binding protein, recently identified as an essential regulator of DNA replication in Helicobacter pylori. We report the crystal structure of HobA in complex with domains I and II of DnaA (DnaA I-II ) from H. pylori, the first structure of DnaA bound to one of its regulators. Biochemical characterization of the complex formed shows that a tetramer of HobA binds four DnaA I-II molecules, and that DnaA I-II is unable to oligomerize by itself. Mutagenesis and protein-protein interaction studies demonstrate that some of the residues located at the HobA-DnaA I-II interface in the structure are necessary for complex formation. Introduction of selected mutations into H. pylori shows that the disruption of the interaction between HobA and DnaA is lethal for the bacteria. Remarkably, the DnaA binding site of HobA is conserved in DiaA from Escherichia coli, suggesting that the structure of the HobA/ DnaA complex represents a model for DnaA regulation in other Gram-negative bacteria. Our data, together with those from other studies, indicate that HobA could play a crucial scaffolding role during the initiation of replication in H. pylori by organizing the first step of DnaA oligomerization and attachment to oriC.DiaA ͉ isothermal titration calorimetry ͉ replication regulators ͉ X-ray crystallography

Regulating DNA Replication in Bacteria

“…In addition, the HobA protein of Helicobacter pylori (Hp), a member of 1-Proteobacteria, displays functional and structural similarity to DiaA, although there is no significant sequence similarity (Table 1) (Natrajan et al 2007(Natrajan et al , 2009Zakrzewsak-Czerwinska et al 2007;Zawilak-Pawlik et al 2007, 2011Terradot et al 2010). …”

The Sporulation Protein SirA Inhibits the Binding of DnaA to the Origin of Replication by Contacting a Patch of Clustered Amino Acids

“…Interestingly, DiaA and HobA interact with DnaA at residues corresponding to those we have identified as the binding site for SirA (13,20). This is the case even though these three proteins alter DnaA function in different ways.…”

“…Domain I is also the contact site for two regulators of DnaA in Gram-negative bacteria that promote replication initiation. These are DiaA from E. coli (13) and HobA from Helicobacter pylori (20). DiaA interacts with DnaA in a way that increases the initiator's affinity for DnaA boxes, resulting in more DnaA binding to the origin and faster initiation (14).…”

“…DiaA interacts with DnaA in a way that increases the initiator's affinity for DnaA boxes, resulting in more DnaA binding to the origin and faster initiation (14). HobA, which is essential in H. pylori, dimerizes DnaA domain I monomers (20). H. pylori DnaA lacks the W6 residue that is essential for dimerization of this domain in E. coli (1), and it is thought that HobA is required to replace this function.…”

Structural insight intoHelicobacter pyloriDNA replication initiation

“…19 The structure of the HobA/ DnaA I-II complex contains a tetramer of HobA and four molecules of DnaA I-II located at opposite poles of the HobA tetramers. (Fig.…”