3bos

Protein Summary

Structural Basis of Regulatory Inactivation of DnaA

Regulatory inactivation of DnaA (RIDA) is an important mechanism for regulating the activity of DnaA to prevent over initiation during bacterial chromosomal replication. RIDA is dependent on protein Hda and the sliding clamp of DNA polymerase III. Hda protein, when bound to the sliding clamp loaded on the duplex DNA, can trigger the transformation of DnaA from the active DnaA-ATP form into the inactive DnaA-ADP form. Hda is homologous to the ATPase region of DnaA and a member of the family of AAA+ ATPases. The crystal structure of dimeric Hda protein from Shewanella Amazonensis SB2B was determined to 1.75 Å by high-throughput protein crystallography. It contains two domains (1-174, 175-241) which correspond to domain III and domain IIIb of DnaA. The arrangement of the two domains in Hda differs dramatically from that of DnaA. Two sliding clamp binding motifs at the N-terminus (13-18) form an antiparallel beta-sheet at the dimer interface and are located on the same face of the dimer. The dimer of Hda demonstrates a new mode of oligomeric assembly of AAA+ proteins in which the two arginine fingers (Arg 161) crucial for the inactivation of DnaA-ATP are fully exposed. One cytidine diphosphate (CDP) molecule was found in the active site of each Hda monomer. CDP is perfered over ADP/UDP by the conserved binding site. This raises the possibility of Hda activity being regulated by CDP (or CTP). Analysis of Walker A and B motifs of Hda  proteins suggests that Hda may have lost its NTPase acitivity. The structure of Hda provides us valuable insights into the regulation of Hda protein as well as the interactions between Hda and the sliding clamp and DnaA.