Defining the solution state dimer structure of Escherichia coli SecA using Förster resonance energy transfer.

Nearly one third of the proteins synthesized in the cytoplasm of bacteria must either insert into or cross the plasma membrane in order to reach their functional location within the cell. In Escherichia coli, the General Secretion pathway (Sec pathway) is the major pathway for secretory protein translocation (reviewed in du Plessis et al.1). The Sec machinery consists of two major components, a heterotrimeric translocon complex, composed of SecY, SecE, and SecG, that is located within the plasma membrane, and a large soluble ATPase protein, SecA, that binds preproteins and associates with SecYEG. At this latter location, SecA utilizes ATP-dependent conformational changes to drive transport of preproteins through the protein-conducting channel of SecYEG. The oligomeric form of both SecA and SecYEG in their functional state has remained a matter of considerable controversy during the past decade, as has the precise structural form of the different dimer states observed in vitro (for a recent review, see Rusch et al.2). E. coli SecA protein was originally described as a homodimer in solution that is subject to a concentration-dependent monomer-dimer equilibrium which can be modulated by both temperature and salt concentration 3-5. This observation was followed up by numerous reports that described how key SecA ligands such as nucleotides, signal peptides, phospholipids, and SecYEG, all affect its dynamic monomer-dimer equilibrium (for a recent review, see 6). Such findings have been used to argue that SecA functions as either a monomer or a dimer or in some type of monomer-dimer cycle during the normal protein translocation cycle. Deficiencies with the current equivocal literature fall along several main lines: (i) certain studies utilized non-equilibrium techniques that can distort the normal monomer-dimer equilibrium, such as protein crosslinking, to assess SecA monomer-dimer status 7, 8, (ii) other studies utilized monomer-biased SecA or preactivated SecY (prlA4) mutants, duplicated and linked gene copies (e.g. SecAA or SecYY), or artificially crosslinked dimers that can give rise to artificial situations 8-15, (iii) the presence of detergents or high salt in biochemical or structural studies of the SecA-SecYEG complex would induce SecA monomerization 16, 17, (iv) certain biochemical assays were not sensitive enough to detect residual SecA dimer, while other assays utilized potentially non-physiological levels of ligands such as signal peptides that could have resulted in nonspecific effects 7, 18, 19. Clearly the use of additional in vivo or equilibrium in vitro methodologies is needed in order to resolve this complex but critical question of SecA oligomer function. In that regard, recent fluorescence burst analyses, an equilibrium technique, indicated that SecA bound to SecYEG as a dimer 20. Not only has the oligomeric functional state of SecA been a matter of dispute, but also the precise structural form of the SecA dimer has remained unclear given the different SecA dimer crystal forms that have been observed recently. SecA from four bacterial species has been crystallized as both monomers and dimers 21-26. While all of the SecA crystal structures displayed a similar protomer building block, strikingly different dimer interfaces were observed (Figure 1). The initial B. subtilis SecA crystal structure (1M6N) predicted an anti-parallel dimer orientation with extensive interactions between the various N-terminal and C-terminal domains of SecA on opposing protomers, which was supported experimentally by a study employing FRET 21, 27. However since then, four additional SecA dimer structures have been published, all with different dimer interfaces. The M. tuberculosis SecA dimer structure (1NL3) also has the two protomers arranged in an antiparallel fashion, but it contains the smallest dimer interface formed by symmetrical interactions between two α-helices at the junction of NBF21 and HSD of one protomer with the tip of the two-helix finger sub-domain and a β-strand of PPXD of the other protomer 23. An alternative antiparallel B. subtilis SecA dimer structure (2IBM) has the two protomers arranged in an orthogonal fashion utilizing a dimer interface that is located at the prominent groove formed by the junction of NBF2 and PPXD 25. The E. coli SecA dimer structure (2FSF) also has the two protomers arranged in an antiparallel fashion, but the dimer interface consists solely of contacts between NBF1 and NBF2 of the respective protomers 22. Finally, the T. thermophilus SecA dimer structure (2IPC) is the only one presently that has the two protomers arranged in a parallel fashion in which an α-helix from NBF2 and the most N-terminal portion of HSD of one protomer insert into the NBF1-NBF2 interface of the other protomer to form a hinge-like structure that gives the dimer an overall open scissors-like configuration along its long axis 24. Whether any of these SecA dimers is physiologically relevant or is solely the result of its crystallization conditions remains presently unclear. In that regard, a recent cryo-electron microscopy study supported an antiparallel orientation for the E. coli SecA dimer, but it found a more extensive dimer interface that was incompatible with the E. coli SecA crystal structure 28. Figure 1 SecA dimer crystal structures with monocysteine residues colored. The color assignments for the residues are as follows: 59-dark green, 340-yellow, 402-blue, 427-hot pink, 458-brown, 470-purple, 506-red, 696-green, and 734-orange. E. coli amino acid residues ... The existence of additional SecA dimer structures has led to problems in interpreting the relevant literature. For example, the small data set obtained in the FRET study of Ding et al. 27 that was originally utilized to support the 1M6N dimer orientation is now inadequate given its potential compatibility with other SecA dimers. Likewise, the crosslinking study of Jilaveanu et al. 13 was consistent with the 1M6N dimer, but it is compatible with the 2IPC dimer as well 10. Finally, the behavior of the monomer-biased SecAΔ11 mutant that is missing N-terminal residues 2-11 was consistent with the 1M6N dimer, since this region is an important subunit contact site for this structure 8, 11; however this region also plays a similar role for the 2IPC dimer 24. Given these ambiguities, additional studies are now needed in order to clearly distinguish between the different SecA dimer forms and determine which one(s) is physiologically relevant for SecA in solution or bound to its different ligands. Considerable information on the structure and function of SecA has been gained through the use of fluorescence techniques. Previous fluorescence studies of SecA have utilized intrinsic tryptophan fluorescence along with collisional quenchers to study SecA conformational changes mediated by temperature or binding of signal peptides, nucleotides, model membranes, or SecYEG-containing proteoliposomes 18, 29-31. FRET approaches have been used successfully to study conformational changes between PPXD and HSD of SecA that are induced by the binding of nucleotide or model membranes, as well as to assess the dimeric form of SecA in solution and to study its stability in the presence of phospholipids, signal peptides, and nucleotides 7, 27, 32. More recently, FRET has been used to map the signal peptide-binding site on SecA to the PPXD-NBF1-HSD multi-domain interface 33, which was found to be consistent with earlier biochemical and NMR studies 34-36. Collectively, these studies demonstrate the utility of fluorescence and particularly the FRET methodology for detailed elucidation of SecA structure-function relationships under biochemically relevant and more physiological conditions. In order to begin to resolve the dominant structural form of the E. coli SecA dimer in its ground solution state, we undertook a FRET approach in the present study. Nine different positions on SecA were chosen for dye labeling in order to distinguish between the previously observed SecA dimers, and three different dye pairs with widely different R0 values were utilized to make accurate interprotomer measurements from 20-120 A. Our data strongly support the existence of a 1M6N-like dimer as the dominant form of SecA protein in solution 21. Furthermore, we have also investigated structural changes of the SecA dimer upon signal peptide binding. Our data indicate that SecA dimer structure is maintained upon signal peptide binding, but the PPXD and HWD regions experience significant conformational changes during this interaction. Intriguingly, movement of the PPXD is greatly enhanced by the use of an elongated signal peptide containing an additional 19 residues from the early mature region of the preprotein.