Penetration into membrane of amino‐terminal region of SecA when associated with SecYEG in active complexes

Abstract

The general secretory (Sec) system of Escherichia coli translocates both periplasmic and outer membrane proteins through the cytoplasmic membrane. The pathway through the membrane is provided by a highly conserved translocon, which in E. coli comprises two heterotrimeric integral membrane complexes, SecY, SecE, and SecG (SecYEG), and SecD, SecF, and YajC (SecDF/YajC). SecA is an associated ATPase that is essential to the function of the Sec system. SecA plays two roles, it targets precursors to the translocon with the help of SecB and it provides energy via hydrolysis of ATP. SecA exists both free in the cytoplasm and integrally membrane associated. Here we describe details of association of the amino-terminal region of SecA with membrane. We use site-directed spin labelling and electron paramagnetic resonance spectroscopy to show that when SecA is co-assembled into lipids with SecYEG to yield highly active translocons, the N-terminal region of SecA penetrates the membrane and lies at the interface between the polar and the hydrophobic regions, parallel to the plane of the membrane at a depth of approximately 5 Å. When SecA is bound to SecYEG, preassembled into proteoliposomes, or nonspecifically bound to lipids in the absence of SecYEG, the N-terminal region penetrates more deeply (8 Å). Implications of partitioning of the SecA N-terminal region into lipids on the complex between SecB carrying a precursor and SecA are discussed.

Substrate Proteins Take Shape at an Improved Bacterial Translocon

“…Recently, Koch et al (14) utilized single copies of SecYEG reconstituted into nanodiscs of differing lipid content in order to show that SecA gains access to SecYEG translocons via a lipidbound intermediate utilizing SecA protein's amphipathic lipid-binding N terminus. More recently, Findik et al (15) showed that this region of SecA assumed different conformations in PLYEG and PLYEG•SecA liposomes, where the N terminus penetrated the lipid bilayer more deeply in the former case, while it was found in a more shallow state, parallel to the plane of the membrane at the interface between the polar and hydrophobic regions, in the latter case. These formative studies help to define the role of the SecA N-terminal ϳ20 residues as a molecular switch that alternates between two lipid-inserted conformations that turn off and on its high-affinity SecYEG binding and translocation ATPase activities (16,17).…”

Interaction of the Motor Protein SecA and the Bacterial Protein Translocation Channel SecYEG in the Absence of ATP

“…3 The transition between state I and II may be associated with the insertion of the N-terminus into the lipid membrane as previously suggested. 4 The model does not refer to the SecA dimer, because it is (i) apparently unable to bind to the membrane surface 36,37 , and (ii) dissociates the presence of acidic lipids 25 . Reports about the functionality of covalently linked SecA dimers 5,42 do not challenge the conclusion, because they do not contain evidence that the dimer may bind to the lipid in the absence of SecYEG.…”

“…44 Our model suggests that the insertion of SecA's N-terminus into the lipid membrane hinders a deep penetration of SecA's two-helix finger into SecYEG. Thus, SecA is trapped shuttling in between state IV and V. However, it does not require a major conformational change 2,4 to strongly bind to SecYEG as in state III the N-terminus is functioning solely as membrane-tether 7 that is electrostatically linked to the membrane, but not inserted into it.…”

“…3 The N-terminus thereby penetrates into the membrane and aligns parallel to the membrane-plane at a depth of 7-8 . 4 Deletion of the first 20 amino acids of the N-terminus (SecA His-ΔN20) impedes SecA dimerization. 5,6 The accompanying activity loss can be reversed by substituting the N-terminus for a His-tag and supplementing SecYEG proteoliposomes with Ni + -NTA lipids, suggesting that membrane tethering is important for functioning.…”

“…Thus, a major conformational change of SecA was suggested to allow SecYEG binding and penetration of the N-terminus at the same time. 2,4 Yet, structural evidence for SecA's large conformational change is lacking.…”