Identification of the Preprotein Binding Domain of SecA

Abstract

SecA, the preprotein translocase ATPase, has a helicase DEAD motor. To catalyze protein translocation, SecA possesses two additional flexible domains absent from other helicases. Here we demonstrate that one of these "specificity domains" is a preprotein binding domain (PBD). PBD is essential for viability and protein translocation. PBD mutations do not abrogate the basal enzymatic properties of SecA (nucleotide binding and hydrolysis), nor do they prevent SecA binding to the SecYEG protein conducting channel. However, SecA PBD mutants fail to load preproteins onto SecYEG, and their translocation ATPase activity does not become stimulated by preproteins. Bulb and Stem, the two sterically proximal PBD substructures, are physically separable and have distinct roles. Stem binds signal peptides, whereas the Bulb binds mature preprotein regions as short as 25 amino acids. Binding of signal or mature region peptides or full-length preproteins causes distinct conformational changes to PBD and to the DEAD motor. We propose that (a) PBD is a preprotein receptor and a physical bridge connecting bound preproteins to the DEAD motor, and (b) preproteins control the ATPase cycle via PBD.

Use of synthetic signal sequences to explore the protein export machinery

“…Using fragments, deletional analysis, and chemical crosslinking of synthetic signal peptides, Economou's group concluded that the region at the base of the PPXD (so-called stem region) serves to bind the signal sequence. 69,89 Results of a FRET-based study with complementary crosslinking approaches also implicated the PPXD (specifically the third Using Signal Peptides to Explore Protein Export 315 helix) in signal sequence binding, 93,95 which is consistent with the early crosslinking studies from Mizushima's lab. 94 We have performed a sequence alignment of 550 SecA proteins, and the region proposed by Musial-Siwek et al 93 has a low conservation score (J. L. Maki and L. M. Gierasch, unpublished observations), raising doubt about whether it serves as a direct binding site for signal sequences.…”

“…69 The PPXD is implicated in binding to open SecA by the finding that signal peptides stabilize an isolated construct corresponding to the PPXD in a distinct conformation. 89 As expected for a complex interplay of ligand-modulated conformational transitions, the binding of signal peptides shifts the closed to open equilibrium in SecA and modulates its ATPase activity. The addition of signal peptide to SecA in solution and in phospholipids/liposomes causes a change in SecA fluorescence 79,96 as well as in protease sensitivity.…”

“…75,76,79,80 Thus, the picture that emerges is that the ligand-mediated triggering of domain dissociation leads to insertion of SecA into the membrane and activation of its translocase function. Economou's group 69,89 pointed to a specific region near the C-terminus, residues 783-795, that serves as an intramolecular regulator of ATPase activity (IRA1), and proposed that ligands (or deletion) caused the release of this IRA inhibition. They also identified another region that had synergistic effects, and termed it IRA2 and the first segment IRA1.…”

“…69,72 These results support an interdependent role of IRA1 and signal peptides during the translocation cycle. 69,89 A System to Explore How Signal Peptides Bind the Open Conformation of SecA…”

Cryo-electron Microscopic Structure of SecA Protein Bound to the 70S Ribosome

“…The cleft between the PPXD and NBD2 is referred to as the clamp, and, depending on the position of the PPXD, it can be in an open (5), partially open (8), or closed form (10). The PPXD has been proposed to interact with preproteins (11)(12)(13)(14).…”