Characterization of a polypeptide‐binding site in the DEAD Motor of the SecA ATPase

We coupled peptides from a CNBr digest of signal‐sequenceless maltose‐binding protein (MBP) to a surface plasmon resonance chip. SecA‐N95, SecA‐N68, and SecA‐DM (which consists of only the DEAD Motor domains NBD1 and NBD2) bound to the immobilized peptides; ADP weakened the binding. SecA‐DM, which lacks the ‘preprotein cross‐linking domain’ (PPXD), displayed the most extensive binding, while an MBP‐PPXD chimera showed no binding, demonstrating that the PPXD does not contribute to the binding. We characterized the sequence specificity using oriented peptide libraries; these results enabled synthesis of a 20‐residue peptide that was used to recapitulate the results obtained with MBP‐derived peptides. This study shows that there is a promiscuous and nucleotide‐modulated peptide‐binding site in the DEAD Motor domains of SecA.