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 t he Escherichia coli precursor protein translocation cascade. SecA cont ains two essential nucleotide binding sites (NBSs), i.e., NBS1 and NBS 2 that hind ATP with high and low affinity, respectively. The photoact ivatable bifunctional cross-linking agent 3'-arylazido-8-azidoadenosin e 5'-triphosphate (diN(3)ATP) was used to investigate the spatial arra ngement of the nucleotide binding sites of SecA, DiN(3)ATP is an authe ntic ATP analogue as it supports SecA-dependent precursor protein tran slocation and translocation ATPase, UV-induced photo-cross-linking of the diN(3)ATP-bound SecA results in the formation of stable dimeric sp ecies of SecA. D209N SecA, a mutant unable to bind nucleotides at NBS1 , was also photo-crosslinked by diN(3)ATP, whereas no cross-linking oc curred with the NBS2 mutant R509K SecA, We concluded that the low-affi nity NBS2, which is located in the carboxyl-terminal half of SecA, Is the site of crosslinking and that NBS2 binds nucleotides at or near th e subunit interface of the SecA dimer.