The Long α-Helix of SecA Is Important for the ATPase Coupling of Translocation

Abstract

SecA contains two ATPase folds (NBF1 and NBF2) and other interaction/regulatory domains, all of which are connected by a long helical scaffold domain (HSD) running along the molecule. Here we identified a functionally important and spatially adjacent pair of SecA residues, Arg-642 on HSD and Glu-400 on NBF1. A charge-reversing substitution at either position as well as disulfide tethering of these positions inactivated the translocation activity. Interestingly, however, the translocation-inactive SecA variants fully retained the ability to up-regulate the ATPase in response to a preprotein and the SecYEG translocon. The translocation defect was suppressible by second site alterations at the hinge-forming boundary of NBF2 and HSD. Based on these results, we propose that the motor function of SecA is realized by ligand-activated ATPase engine and its HSD-mediated conversion into the mechanical work of preprotein translocation.

Different modes of SecY–SecA interactions revealed by site-directed in vivo photo-cross-linking

“…On the other hand, the first interaction involving C6 is related to the event, in which SecA actively drives translocation. These interpretations are consistent with our genetic observation that ATPase activation and preprotein translocation are separable (58). The fact that a number of the C6 residues can be cross-linked to SecA might imply that SecA is actually moving along the C6 region of SecY.…”

SecA Drives Transmembrane Insertion of RodZ, an Unusual Single-Span Membrane Protein

“…As a final test of the SecA requirement, we used a dominant-negative SecA system. We co-expressed RodZ with the dominant-negative SecA(R642E) under T7-promoter control by transforming BL21 cells with the pET21-T7-RodZ-SecA(R642E) vector [34]. Fig.…”

Mapping of the SecA·SecY and SecA·SecG Interfaces by Site-directed in Vivo Photocross-linking

“…In that regard the two SecG-reactive SecA residues, 402 and 647, are adjacent to Glu-400 and Arg-642, respectively. These latter two residues form an ionic pair that regulates coupling between SecA ATPase and protein translocation cycles (56). Association of SecG with this region of SecA may help to destabilize this ionic pair, thereby releasing an inhibitory interaction between the SecA DEAD motor and its proposed two-helix finger ratchet within the HSD domain of SecA (38,57).…”