Two distinct ATP-binding domains are needed to promote protein export by Escherichia coli SecA ATPase.

Six putative ATP-binding motifs of SecA protein were altered by oligonucleotide-directed mutagenesis to try to define the ATP-binding regions of this multifunctional protein. The effects of the mutations were analysed by genetic and biochemical assays. The results show that SecA contains two essential ATP-binding domains. One domain is responsible for high-affinity ATP binding and contains motifs A0 and B0, located at amino acid residues 102-109 and 198-210, respectively. A second domain is responsible for low-affinity ATP binding and contains motifs A3 and a predicted B motif located at amino acid residues 503-511 and 631-653, respectively. The ATP-binding properties of both domains were essential for SecA-dependent translocation ATPase and in vitro protein translocation activities. The significance of these findings for the mechanism of SecA-dependent protein translocation is discussed.