Figure 3. Effects of ATP and ADP on the nucleotide binding site.
(A) Closeup of the NBS of SecA in the T. maritima SecYEG-SecA structure (3DIN; [Zimmer et al., 2008]).The ADP-BeFx moiety is shown as sticks (carbon green, nitrogen blue, oxygen red, phosphorus orange, beryllium lime and fluorine pale blue), and key helicase loops are shown as white cartoons. Conserved helicase features are shown as coloured sticks and numbered as in T. maritima: Glu99 of Walker A (helicase motif Ia); Glu253 of Walker B (DEAD box of helicase motif II); Thr416 of helicase motif III; and Arg574 (‘arginine finger’) of helicase motif VI. A black dotted line marks the distance between Arg574 and Glu99. (B) The same view as in panel A, but for post-microsecond simulation snapshots in the open, part-open and closed simulations. Of note are Arg574 and Glu99, which remain distant in the open and part-open simulations (ATP), but come close enough to form a salt bridge in the closed simulation (ADP). In helicases, this conserved arginine finger on motif VI has been shown to couple ATPase activity with substrate binding (Hall et al., 1998), underlining its importance in SecA. After the Arg574 to Glu99 salt bridge has formed in the ADP simulations, the Walker A motif is bought into contact with a conserved threonine (Thr416) in helicase motif III, which in the helicase family has been shown to use ATP binding to create an RNA binding site (Banroques et al., 2010). This threonine communicates directly with the SecA DEAD box—providing a likely route forsignal transmission of ATP hydrolysis away from the NBS towards the polypeptide binding sites. (C) Minimum distance analysis between residues Glu99 and Arg574 of SecA across the simulation data confirms that a Glu99-Arg574 interaction is formed in 4/5 of the ADP simulations, whereas it is absent in all 5 ATP simulations. Interestingly, the ADP simulation that does not find this salt bridge is the same simulation in which the SecY LG samples the part-open state (Figure 2C). Thus, it appears that nucleotide dependent formation and breaking of the Glu99-Arg574 salt bridge is involved in the coupling process leading ultimately to the respective closure and opening of the channel.