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

Abstract

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.

The ATPase domain of SecA can form a tetramer in solution 1 1Edited by I. B. Holland

“…38,39 However, the presence of the N68 tetramer demonstrates that the N-terminal ATPase domain has the ability to mediate protomer-protomer interactions: these same interactions may occur within a SecA dimer. Given that the nucleotide, preprotein, and SecY binding sites reside on N68, 34,35,37 direct interactions between the N-terminal ATPase domains of the SecA molecule may be important in the regulation and catalysis of translocation.…”

“…The SecA protomer can be divided into two domains: an N-terminal domain, from residues 1-610, and a C-terminal domain, from 610 to 901. 30 The N-terminal ATPase domain contains seven consensus DEAD helicase motifs, 31,32 including the Walker A and B motifs 33 that are critical for high af®nity nucleotide binding and hydrolysis, 34 and it also contains a preprotein cross-linking site. 35 The N-terminal domain is able to bind peptides corresponding to signal sequences, and this binding decreases the intrinsic ATPase activity of the domain.…”

The Sec system

“…SecA contains two essential nucleotide-binding sites (NBSs) [20,21] and undergoes large nucleotide-induced conformational changes during translocation (reviewed in [1"]). The thermal unfolding characteristics of SecA suggest the presence of at least two independent folding domains of near equal size [22"].…”

Evidence that KpsT, the ATP-binding component of an ATP-binding cassette transporter, is exposed to the periplasm and associates with polymer during translocation of the polysialic acid capsule of Escherichia coli K1

“…Consistent with this general model are recent studies of SecA that have more fully elucidated the role of ATP binding and hydrolysis in promoting insertion of SecA into the membrane and deinsertion. A mutation in D209 (analogous to D149 in motif B of KpsT) preserves ATP binding by SecA, albeit at a depressed level, but eliminates the protein's translocation ATPase activity (14). This protein, defective in protein translocation, was shown to be stably inserted into the membrane but defective in deinsertion (7).…”