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The exceptionally tight affinity of DnaA for ATP/ADP requires a unique aspartic acid residue in the AAA+ sensor 1 motif
Author(s) -
Kawakami Hironori,
Ozaki Shogo,
Suzuki Shigeo,
Nakamura Kenta,
Senriuchi Takayuki,
Su'etsugu Masayuki,
Fujimitsu Kazuyuki,
Katayama Tsutomu
Publication year - 2006
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2006.05450.x
Subject(s) - dnaa , dnab helicase , biology , biochemistry , atp hydrolysis , nucleotide , dna binding protein , mutant , dna , atpase , microbiology and biotechnology , dna replication , helicase , enzyme , rna , origin of replication , gene , transcription factor
Summary 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.