1. The exceptionally tight affinity of DnaA for ATP/ADP requires a unique aspartic acid residue in the AAA+ sensor 1 motif.
- Author
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Kawakami H, Ozaki S, Suzuki S, Nakamura K, Senriuchi T, Su'etsugu M, Fujimitsu K, and Katayama T
- Subjects
- Amino Acid Motifs, Bacterial Proteins chemistry, Bacterial Proteins genetics, Binding Sites, DNA-Binding Proteins chemistry, DNA-Binding Proteins genetics, Escherichia coli genetics, Escherichia coli growth & development, Adenosine Diphosphate metabolism, Adenosine Triphosphate metabolism, Aspartic Acid chemistry, Bacterial Proteins metabolism, DNA-Binding Proteins metabolism, Escherichia coli metabolism
- Abstract
Escherichia coli DnaA, an AAA+ superfamily protein, initiates chromosomal replication in an ATP-binding-dependent manner. Although DnaA has conserved Walker A/B motifs, it binds adenine nucleotides 10- to 100-fold more tightly than do many other AAA+ proteins. This study shows that the DnaA Asp-269 residue, located in the sensor 1 motif, plays a specific role in supporting high-affinity ATP/ADP binding. The affinity of the DnaA D269A mutant for ATP/ADP is at least 10- to 100-fold reduced compared with that of the wild-type and DnaA R270A proteins. In contrast, the abilities of DnaA D269A to bind a typical DnaA box, unwind oriC duplex in the presence of elevated concentrations of ATP, load DnaB onto DNA and support minichromosomal replication in a reconstituted system are retained. Whereas the acidic Asp residue is highly conserved among eubacterial DnaA homologues, the corresponding residue in many other AAA+ proteins is Asn/Thr and in some AAA+ proteins these neutral residues are essential for ATP hydrolysis but not ATP binding. As the intrinsic ATPase activity of DnaA is extremely weak, this study reveals a novel and specific function for the sensor 1 motif in tight ATP/ADP binding, one that depends on the alternate key residue Asp.
- Published
- 2006
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