The Low-Affinity ATP Binding Site of theEscherichia coliSecA Dimer Is Localized at the Subunit Interface

Abstract

The homodimeric SecA protein is the ATP-dependent force generator in the Escherichia coli precursor protein translocation cascade. SecA contains two essential nucleotide binding sites (NBSs), i.e., NBS1 and NBS2 that bind ATP with high and low affinity, respectively. The photoactivatable bifunctional cross-linking agent 3‘-arylazido-8-azidoadenosine 5‘-triphosphate (diN3ATP) was used to investigate the spatial arrangement of the nucleotide binding sites of SecA. DiN3ATP is an authentic ATP analogue as it supports SecA-dependent precursor protein translocation and translocation ATPase. UV-induced photo-cross-linking of the diN3ATP-bound SecA results in the formation of stable dimeric species of SecA. D209N SecA, a mutant unable to bind nucleotides at NBS1, was also photo-cross-linked by diN3ATP, whereas no cross-linking occurred with the NBS2 mutant R509K SecA. We concluded that the low-affinity NBS2, which is located in the carboxyl-terminal half of SecA, is the site of cross-linking and that NBS2 binds nucleotides at or near the subunit interface of the SecA dimer.

SecAAA trimer is fully functional as SecAA dimer in the membrane: Existence of higher oligomers?

“…It is known that SecA can be crosslinked as dimers, or even higher order oligomers in vitro [17,22,35–37]. Parts of the controversy of whether Sec functions as dimers were the contradictory observations that depending on the cross-linked conditions, some cross-linked SecA dimers were active [36, 38] where the other were not [21].…”

The bacterial translocase: a dynamic protein channel complex

“…NBS-II was mapped at a more C-terminal position than NBS-I. The use of bifunctional photo-activatable ATP analogs indicates that NBS-II is located at or near the subunit interface of the SecA dimer [87]. The structures of Mg 2+ -ADP-bound B. subtilis SecA and Mg 2+ -ADP-b-Sbound M. tuberculosis SecA1, however, lack indications for the binding of a second nucleotide [83,84].…”

Thermodynamics of nucleotide binding to NBS‐I of the Bacillus subtilis preprotein translocase subunit SecA

“…Translocation of preprotein segments may occur by co‐insertion with SecA domains into the membrane. SecA has two essential nucleotide binding sites (NBSs) [7–11]: a high affinity binding site ( K d ≈0.15 μM) is confined to the amino‐terminal domain of the protein (NBS‐I) and a low affinity binding site ( K d ≈340 μM) is located in the second half of the protein (NBS‐II) near the subunit interface [12]. The functional role of the separate NBSs is still largely unresolved, but the binding of ATP to NBS‐I suffices to insert SecA domains into the membrane [6].…”