Probing the Affinity of SecA for Signal Peptide in Different Environments

Abstract

SecA, the peripheral subunit of the Escherichia coli preprotein translocase, interacts with a number of ligands during export, including signal peptides, membrane phospholipids, and nucleotides. Using fluorescence resonance energy transfer (FRET), we studied the interactions of wild-type (WT) and mutant SecAs with IAEDANS-labeled signal peptide, and how these interactions are modified in the presence of other transport ligands. We find that residues on the third α-helix in the preprotein cross-linking domain (PPXD) are important for the interaction of SecA and signal peptide. For SecA in aqueous solution, saturation binding data using FRET analysis fit a single-site binding model and yielded a K d of 2.4 μM. FRET is inhibited for SecA in lipid vesicles relative to that in aqueous solution at a low signal peptide concentration. The sigmoidal nature of the binding curve suggests that SecA in lipids has two conformational states; our results do not support different oligomeric states of SecA. Using native gel electrophoresis, we establish signal peptide-induced SecA monomerization in both aqueous solution and lipid vesicles. Whereas the affinity of SecA for signal peptide in an aqueous environment is unaffected by temperature or the presence of nucleotides, in lipids the affinity decreases in the presence of ADP or AMP-PCP but increases at higher temperature. The latter finding is consistent with SecA existing in an elongated form while inserting the signal peptide into membranes.More than one-third of the proteins synthesized inside the cell must be exported to extracytoplasmic locations to perform their functions. In Escherichia coli, many preproteins are recognized and transported by the Sec transport machinery. This secretory pathway has been extensively studied and several of the key proteins involved have been identified and characterized; however, the mechanisms by which the preprotein interacts with the secretion machinery are not clearly understood.In the cytoplasm, SecB, a chaperone, binds preproteins to keep them in an unfolded state and delivers them to the membrane-associated SecA for post-translational export (1, 2). SecA is a critical component of the Sec transport pathway; it recognizes and binds the preprotein and functions as an ATPase. Moreover, conformational changes resulting from the interaction of SecB with SecA are thought to result in the transfer of the preprotein from the chaperone to the ATPase (3, 4). The membrane proteins, SecG, SecD, and SecF, stabilize and stimulate SecA at the membrane, and as a consequence, SecA can deliver the preprotein through the SecYEG pore (5, 6). Some studies indicate binding of ATP causes the dissociation of SecB from the enzyme, and cycles of ATP hydrolysis (7,8) and conformational changes lead to membrane insertion of the SecA-preprotein complex followed by deinsertion of SecA (9, 10). Meyer et al. (11) One SecA dimerization site is located at its C-terminus (18), which also binds SecY, SecB, and phospholipids (23), and not surprisingly, these a...

Characterization of the Escherichia coli SecA Signal Peptide-Binding Site

“…Wild-type SecA protein bound PhoA signal peptide with an affinity of 2.4 Ϯ 0.8 M, in agreement with previously published values ( Table 2; see also Fig. S1 in the supplemental material) (2,3,31). Most of our SecA mutant collection had signal peptidebinding affinity constants that were within 3-fold of wild-type SecA, indicating that the relevant substitution had a modest effect or no effect on SecA signal peptide binding.…”

“…A number of studies have utilized genetically mutated, truncated, or proteolytically cleaved SecA proteins along with signal peptide-binding or cross-linking assays to map portions of the relevant ligandbinding site on SecA (3,31,32,37). At best, such approaches are of limited utility because either they identify a small number of residues that may or may not directly contribute to signal peptide binding or they facilitate construction of a linear rather than three-dimensional map of the actual binding site since they also include irrelevant regions and exclude relevant ones.…”

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.…”

In Vivo Membrane Topology of Escherichia coli SecA ATPase Reveals Extensive Periplasmic Exposure of Multiple Functionally Important Domains Clustering on One Face of SecA

“…In this model PPXD and NBF-II would serve to gate the active and inactive channels of the SecYEG dimer, respectively. This suggestion is consistent with the observed extensive MPB labeling pattern of these two comparably sized domains of SecA and also with their observed functions as preprotein binding and SecA-SecYEG regulatory domains, respectively (51,52,(55)(56)(57). In this context PPXD and NBF-II labeling could occur on the same protomer or be divided between different protomers, depending on the SecA oligomeric state (monomer or dimer) as well as the SecYEG dimer orientation (front-to-front or back-to-back (29)).…”

“…This result is consistent with the proposed role of PPXD as the preproteinbinding domain of SecA that transfers bound preprotein to SecYEG (51)(52)(53). Both the 219 -244 and 292-319 regions of SecA, which have been proposed to be critical for signal peptide binding (54,55), contain M/S-labeled residues. NBF-II serves a regulatory role in SecA by controlling the ATPase cycle of NBF-I, which in turn controls SecA membrane cycling (22, 56 -58).…”