Using a Low Denaturant Model To Explore the Conformational Features of Translocation-Active SecA

Protein secretion is an essential process in all forms of life. In gram-negative bacteria, newly synthesized proteins destined for integration into membranes or secretion into the extracellular milieu predominantly traverse the secretory (Sec) pathway (1, 2). Most pre-proteins are targeted to the Sec pathway by a cleavable N-terminal signal sequence and by the peripheral membrane motor protein, SecA. E. coli SecA is a large, dynamic 102 kDa protein that forms homodimers and interacts with many different players during the translocation cycle, including pre-proteins, SecB, membrane, and the SecYEG translocon (3). The cytosolic chaperone SecB binds to a subset of pre-proteins, keeping them in a translocation-competent state. The pre-protein/SecB complex then interacts with SecA, and is localized to the translocon (2). The association of the pre-protein/SecB/SecA ternary complex with SecYEG induces a conformational change in SecA while ATP binding results in SecB release and initiation of translocation (1). The energy derived from ATP hydrolysis by SecA and the proton motive force subsequently drive the translocation of the pre-protein into the periplasmic space (4–6). SecA is a multi-domain protein (Figure 1) with two tandem ATP-binding domains belonging to the DEAD-helicase superfamily, nucleotide-binding fold I (NBF I) and nucleotide-binding fold II (NBF II) (7). At the interface between NBF I and II is the nucleotide-binding cleft, and both NBF I and NBF II contain helicase motifs needed for ATP hydrolysis (7). NBF II, also known as IRA2 (8), undergoes a disorder-order transition during the ATP catalytic cycle (9) and has higher B factors than NBF I in a crystal structure of SecA from B. subtilis (7). A domain not found in other DEAD-helicases is the pre-protein cross-linking domain (PPXD), which interrupts NBF I (10). Two fragments of SecA can be individually expressed in E. coli or isolated by proteolytic cleavage: N68, a stable 68 kDa fragment of SecA comprised of NBF I, NBF II, and PPXD, and C34, formed by the α-helical scaffold domain (HSD), the α-helical wing domain (HWD), and the C-terminal linker (CTL) (11). The CTL region of the molecule, which includes a zinc-binding motif, contains the SecB-binding site and is also proposed to interact with anionic phospholipids (12). The ATPase activity of cytosolic SecA is suppressed by a helix-loop-helix motif in the HSD, called the IRA1 (11), and is positively regulated by NBF II (8). Therefore, C-terminally truncated constructs of SecA such as N68 (11) and SecA64 (13) possess elevated and unregulated ATPase activity. Figure 1 Domain organization and structural plasticity of the SecA PPXD. The overall domain organization of SecA in the three different crystal structures is similar, but the orientation of PPXD is different. (A) In the B. subtilis SecA structure (7) (PDB: 1M6N), ... Various translocation components induce conformational changes in SecA during the pre-protein translocation cycle. SecA crystal structures show alterations in the positioning of the PPXD domain in relation to the HWD and NBF II (7, 14–16) (Figure 1). The recent 4.5 A structure of SecYEG-bound SecA (16) shows the PPXD rotating away from HWD and making contact with NBF II (Figure 1C), thus forming a clamp region that is proposed to act as a channel for pre-protein translocation. Moreover, solution studies have suggested that even larger conformational changes may occur in SecA with at least two extreme conformational states: a compact, closed form in cytosolic SecA, and a more open state in translocation-active SecA. While ADP binding (17) and reduced temperature (18) favor the closed conformation, factors such as increased temperature (19), mutations (20), denaturants (21), association with model membranes (22, 23), and binding to SecYEG (24) push SecA into a more open conformation. A complete understanding of the complex mechanism of SecA-mediated protein translocation cycle requires identifying and characterizing the various conformational states of SecA and deducing their roles in the translocation cycle. The most dramatic conformational change is believed to occur in ‘translocation-active SecA’. Generating this state requires the presence of all the components of translocation machinery making it challenging to study. We have used the strategy of mild perturbing the SecA native state in aqueous buffer and exploring how it shifts to populate a higher energy state on its energy landscape (25, 26). Associating properties of the newly populated state with functional characteristics of translocation-active SecA has allowed us to interrogate the conformational features of this elusive state. One of the hallmark features of translocation-active SecA is its enhanced ATPase activity (27), and such an activated state of SecA is reported to stably exist in low concentrations of denaturants such as guanidinium chloride or urea (21). In this study, we have characterized SecA in a low concentration of urea, and our findings provide a compelling model for the conformational transition in SecA that accompanies SecA-membrane/translocon binding and commitment of the pre-secretory complex to move the pre-protein across the membrane. The picture that emerges is that of a delicate balance of intradomain metastability and stabilizing interdomain interactions that are readily destabilized upon interaction with functional partners (membrane lipids, SecB, SecYEG, precursor protein, signal peptide, ATP).