Novel essential residues of Hda for interaction with DnaA in the regulatory inactivation of DnaA: unique roles for Hda AAA Box VI and VII motifs.
Escherichia coli ATP-DnaA initiates chromosomal replication. For preventing extra-initiations, a complex of ADP-Hda and the DNA-loaded replicase clamp promotes DnaA-ATP hydrolysis, yielding inactive ADP-DnaA. However, the Hda-DnaA interaction mode remains unclear except that the Hda Box VII Arg finger (Arg-153) and DnaA sensor II Arg-334 within each AAA(+) domain are crucial for the DnaA-ATP hydrolysis. Here, we demonstrate that direct and functional interaction of ADP-Hda with DnaA requires the Hda residues Ser-152, Phe-118 and Asn-122 as well as Hda Arg-153 and DnaA Arg-334. Structural analyses suggest intermolecular interactions between Hda Ser-152 and DnaA Arg-334 and between Hda Phe-118 and the DnaA Walker B motif region, in addition to an intramolecular interaction between Hda Asn-122 and Arg-153. These interactions likely sustain a specific association of ADP-Hda and DnaA, promoting DnaA-ATP hydrolysis. Consistently, ATP-DnaA and ADP-DnaA interact with the ADP-Hda-DNA-clamp complex with similar affinities. Hda Phe-118 and Asn-122 are contained in the Box VI region, and their hydrophobic and electrostatic features are basically conserved in the corresponding residues of other AAA(+) proteins, suggesting a conserved role for Box VI. These findings indicate novel interaction mechanisms for Hda-DnaA as well as a potentially fundamental mechanism in AAA(+) protein interactions.
Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
Kenta
K
Nakamura
Tsutomu
T
Katayama
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; D000251:Adenosine Triphosphatases; C435211:Hda protein, E coli