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

Hiroyuki Mori,Koreaki Ito

doi:10.1074/jbc.m606906200

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.

Highlights

The crystal structures of the translocation components have provided an important basis for our understanding of the translocation molecular mechanisms, the static structures alone are insufficient to reveal the dynamic processes of translocation facilitation
The ATPase and the translocation domains are connected by a characteristic long ␣-helix, termed helical scaffold domain (HSD; Fig. 1A, black), which is running over the molecule and interacting with all the other subdomains
Importance of Arg-642 on HSD and Its Possible Ionic Partner, Glu-400, on NBF1—The HSD part of E. coli SecA contains a number of evolutionarily conserved residues (Fig. 1B, boldface), which were individually mutated to cysteine

Summary

EXPERIMENTAL PROCEDURES

Escherichia coli Strains—JM109 [22] was used as a host for plasmid engineering. KD1087 (mutD5) [23] was used for plasmid mutagenesis. MM52 (secA51(Ts)) [24] and its araϩ deriv-.

Encoded SecA variant

RESULTS

Cs ϩ

DISCUSSION