The exceptionally tight affinity of DnaA for ATP/ADP requires a unique aspartic acid residue in the AAA+ sensor 1 motif
The exceptionally tight affinity of DnaA for ATP/ADP requires a unique aspartic acid residue in the AAA+ sensor 1 motif
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DOI：
10.1111/j.1365-2958.2006.05450.x
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发表时间：
2006-12-01
影响因子：
3.6
通讯作者：
Katayama, Tsutomu
中科院分区：
文献类型：
--
作者：
Kawakami, Hironori;Ozaki, Shogo;Katayama, Tsutomu
大肠杆菌dna是一种AAA+超家族蛋白，以依赖atp结合的方式启动染色体复制。尽管dna具有保守的Walker A/B基序，但它与腺嘌呤核苷酸的结合比许多其他AAA+蛋白紧密10- 100倍。本研究表明，位于传感器1基序的dna Asp-269残基在支持高亲和力ATP/ADP结合中起特定作用。与野生型和DnaA R270A蛋白相比，DnaA D269A突变体对ATP/ADP的亲和力至少降低了10- 100倍。相比之下，DnaA D269A结合典型的DnaA盒子、在ATP浓度升高的情况下解旋oriC双链、将DnaB装载到DNA上以及在重组系统中支持微型染色体复制的能力得以保留。虽然酸性Asp残基在真菌性dna同源物中高度保守，但在许多其他AAA+蛋白中相应的残基是Asn/Thr，在一些AAA+蛋白中这些中性残基是ATP水解所必需的，但不是ATP结合。由于dna的内在ATP酶活性非常弱，本研究揭示了传感器1基序在ATP/ADP紧密结合中的一种新的特异性功能，这种功能依赖于交替的关键残基Asp。
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.