The low-affinity ATP binding site of the Escherichia coli SecA dimer is localized at the subunit interface.
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 crosslinking and that NBS2 binds nucleotides at or near the subunit interface of the SecA dimer.
van der Wolk
J P
JP
Department of Microbiology, University of Groningen, Haren, The Netherlands.
Boorsma
A
A
Knoche
M
M
Schäfer
H J
HJ
Driessen
A J
AJ
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D001386:Azides / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D001665:Binding Sites; D003432:Cross-Linking Reagents; D019281:Dimerization; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D009711:Nucleotides; D019920:Photoaffinity Labels; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D014466:Ultraviolet Rays
C026397:3'(O)-(3-(N-(4-azido-2-nitrophenyl)amino)propionyl)-8-azidoadenosine 5'-triphosphate; D001386:Azides; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D009711:Nucleotides; D019920:Photoaffinity Labels; D000069816:SEC Translocation Channels; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins