Length matters: Functional flip of the short TatA transmembrane helix

Abstract

The twin arginine translocase (Tat) exports folded proteins across bacterial membranes. The putative pore-forming or membrane-weakening component (TatA_d in B. subtilis) is anchored to the lipid bilayer via an unusually short transmembrane α-helix (TMH), with less than 16 residues. Its tilt angle in different membranes was analyzed under hydrophobic mismatch conditions, using synchrotron radiation circular dichroism and solid-state NMR. Positive mismatch (introduced either by reconstitution in short-chain lipids or by extending the hydrophobic TMH length) increased the helix tilt of the TMH as expected. Negative mismatch (introduced either by reconstitution in long-chain lipids or by shortening the TMH), on the other hand, led to protein aggregation. These data suggest that the TMH of TatA is just about long enough for stable membrane insertion. At the same time, its short length is a crucial factor for successful translocation, as demonstrated here in native membrane vesicles using an in vitro translocation assay. Furthermore, when reconstituted in model membranes with negative spontaneous curvature, the TMH was found to be aligned parallel to the membrane surface. This intrinsic ability of TatA to flip out of the membrane core thus seems to play a key role in its membrane-destabilizing effect during Tat-dependent translocation.

Figure 1

SRCD spectra of TatA_2-45 reconstituted in phosphatidylcholine lipid vesicles with varying membrane thickness. All SRCD spectra show a typical α-helical line shape with characteristic bands at 194, 209, and 223 nm. The spectrum of TatA_2-45 in DNPC shows an overall decrease in signal intensity, suggesting some degree of aggregation.

Figure 2

¹⁵N-NMR and SROCD spectra of TatA_2-45 reconstituted in macroscopically aligned phosphatidylcholine lipid bilayers with varying membrane thickness. (A) The ¹⁵N-NMR spectra show that the TMH of TatA_2-45 has an almost upright orientation in the series from DLPC, DMPC, DOPC, and DEiPC, as reflected by distinct signal intensity in the range of 170–220 ppm (highlighted in yellow). With decreasing membrane thickness, these signals shift gradually upfield, indicating a more tilted alignment of the TMH. Pure powder spectra are observed in the case of DNPC and largely in DErPC, as a result of protein aggregation in these very thick bilayers. (B) According to the SROCD line shapes, the TMH of TatA_2-45 possesses an upright orientation in DLPC and DMPC. This is indicated by positive ellipticity values at 208 nm. A strong tilt of the TMH and a flat surface-aligned APH is observed in very short lipids (DOcPC and DDPC) with a strong negative signal at 208 nm. The increasing absorption flattening in DOPC to DNPC, together with an increase of the negative band at 208 nm, indicates aggregation of helical segments. In the SROCD samples, protein aggregation already seems to set in in somewhat thinner membranes than in ¹⁵N-NMR samples. The global minima of the OCD spectra are scaled to the same intensity to allow comparing the different line shapes at the characteristic band around 208 nm (dashed line). A cartoon representation of TatA_2-45 visualizes the qualitative global orientation of the TMH in the membrane, as derived from our NMR/OCD spectra. The hydrophobic membrane thickness (d_c) is given for each lipid (see Table 2).

Figure 3

¹⁵N-NMR and SROCD spectra of TatA_2-45 variants with an extended or shortened TMH reconstituted in macroscopically aligned lipid bilayers composed of DMPC. (A) The signals of the TMH (highlighted in yellow) gradually shift upfield with increasing TMH length, indicating that the TMHs of the variants with an extended TMH become tilted more and more due to the hydrophobic mismatch. The shortened TatA_2-45 ΔLIL shows a high powder content, indicating protein aggregation, because the TMH is too short to span the lipid bilayer. (B) Within the series of the SROCD spectra, the negative band at 208 nm increases with increasing TMH length, indicating a more tilted TMH alignment of the TatA_2-45 variants with an extended TMH (and also the APHs become less and less pulled into the membrane in these variants). A reduction of the signal intensity at around 196 nm is also observed with increasing TMH length (starting from TatA LALAL), indicating some amount of protein aggregation. The TatA_2-45 ΔLIL mutant seems to aggregate, but retains its helical orientation, indicated by a strong reduction in the signal intensity around 194 nm together with a pronounced band at 208 nm. The global minima of the spectra are scaled to the same intensity to allow comparison of the different line shapes at the characteristic band at 208 nm (dashed line). A cartoon representation of TatA_2-45 visualizes the qualitative global orientation of the TMH in the membrane, as derived from our NMR/OCD spectra. The calculated TMH length for each TatA_2-45 variant (see Table 3) and the hydrophobic membrane thickness (d_c) of DMPC lipid bilayers (see Table 2) are given.

Figure 4

¹⁵N-NMR and SROCD spectra of TatA_2-45 variants with varying N-terminal charge density, reconstituted in macroscopically aligned lipid bilayers composed of DOPC, DEiPC, and DErPC. Inspection of the ¹⁵N-NMR spectra shows that the introduction of charged amino acids near the N-terminus leads to an increased tendency of protein aggregation, which sets in already in thinner membranes. In DOPC membranes TatA_2-45 and TatA_2-45 F2D are still well oriented, whereas TatA_2-45 F2DI7D is already aggregated. In the longer DEiPC membranes, only TatA_2-45 is still oriented (highlighted in yellow) and, in the even longer DErPC membranes, all proteins aggregate. A cartoon representation of TatA_2-45 visualizes the qualitative global orientation of the TMH in the membrane, as derived from our NMR spectra (red dots represent the charged N-terminus or the aspartate mutations). To see this figure in color, go online.

Figure 5

¹⁵N-NMR and SROCD spectra of TatA_2-45 reconstituted in oriented lipid bilayers composed of phytanoyl lipids with a highly negative spontaneous curvature. (A) The ¹⁵N-NMR spectra show that phytanoyl lipids support a transmembrane inserted state of the TMH only for the variants with an extended TMH (TatA_2-45 LAL, TatA_2-45 LALAL, and TatA_2-45 LALALAL). The wild-type TatA_2-45 shows a reduced signal of the TMH, suggesting that it flips at least partially out of the membrane core. The mutant TatA_2-45 ΔLIL with a shortened TMH gives no signal of the TMH anymore, but an underlying powder pattern becomes visible. (B) SROCD shows that the TMH of the variants TatA_2-45 LAL and TatA_2-45 LALAL assume a transmembrane inserted state. The mutant TatA_2-45 LALALAL exhibits an increasing negative band at 208 nm, indicating a tilted orientation of the TMH due to positive hydrophobic mismatch (and a flatter surface-aligned APH). The wild-type TMH, as well as the shortened TMH in TatA_2-45 ΔLIL, seem to flip onto the membrane surface, as indicated by the strong negative band at 208 nm. The TatA_2-45 ΔLIL mutant not only flips onto the membrane surface, but partly also aggregates there, as indicated by some amount of absorption flattening. A cartoon representation of TatA_2-45 visualizes the qualitative global orientation of the TMH in the membrane, as derived from our NMR/OCD spectra.

Figure 6

MC simulation of TatA_1-45 with a deprotonated and protonated N-terminus in an implicit membrane model. Snapshot of all-atom MC simulations of TatA_1-45 in an implicit membrane model with a hydrophobic bilayer thickness of 42 Å to examine the role of the N-terminal protonation state and to reveal the structure of the surface-aligned TMH. (A) The deprotonated TMH is stably inserted inside the membrane during the whole simulation. (B) When protonated, the N-terminal helix flips toward the membrane surface and remains there adopting a “banana-shaped” structure, which is kinked in a way that the charged N-terminus and the polar residues point into the hydrophilic environment (see Video S1). To see this figure in color, go online.

Figure 7

In vitro translocation assay of TatA variants with an extended TMH. (A) The presence of TatA, TatB, and TatC in the INVs is monitored using western blots with the corresponding antibodies. In all INVs sufficient amounts of the Tat components were present. However, the expression rate of the TatA variants with a massively extended TMH was slightly reduced. (B) The transport of the natural E. coli Tat substrate AmiC into INVs was monitored. Radioactively labeled AmiC was synthesized by means of in vitro transcription/translation in the presence or absence of INVs. After PK treatment to digest all untransported substrate, only AmiC that has been transported into the lumen of the INVs is detectable in the subsequent SDS-PAGE analysis. A transport efficiency (TE) was quantified on the basis of three independent experiments by comparison of the synthesized amount of AmiC with the amount that has been transported into the lumen. INVs containing the wild-type Tat components (TatA wt) show considerable transport into the vesicles (TE = 35%). It is seen that the transport efficiency is gradually reduced with increasing TMH length (TatA LAL = 16%; TatA LALA = 3%; TatA LALALAL = 1%). This observation proves that the unusually short length of the TMH of TatA plays an important role in the translocation process.

Figure 8

Possible flipping mechanism of the TatA TMH. Cartoon representation of TatA_2-45 with its membrane-spanning TMH (yellow) and its surface-bound APH (blue-yellow). (A) In the case of a flexible hinge, the TatA TMH could swing out of the membrane core. (B) In the case of a rigid linker region, the TatA TMH has to rotate out of the membrane core (view from the cytoplasmic leaflet). To see this figure in color, go online.

Figure 9

Speculative model of TatA TMH flipping triggered by TatC. Cartoon representation of TatA_2-45 with its hydrophobic TMH (yellow) and its amphiphilic APH (blue-yellow). At the start, the TMH of TatA is inserted in an upright transmembrane state, with both the unprotonated N-terminus and the APH immersed sufficiently deep into the membrane core to just about tolerate the hydrophobic mismatch (left panel). Interaction of TatA with its binding site on TatC (indicated by the squiggly line) leads to protonation of the N-terminus (indicated by red dots). The increased polarity leads to stronger hydrophobic mismatch (indicated by red flashes) either at the extracellular face (upper panel) or on the cytoplastic face (lower panel) or both. This strain results in flipping of the protonated TMH toward the cytoplasmic membrane surface (right panel), leading to local membrane weakening and/or permeation, especially if several TatA molecules participate. To see this figure in color, go online.