Lipids trigger a conformational switch that regulates signal recognition particle (SRP)-mediated protein targeting

Abstract

Co-translational protein targeting to the membrane is mediated by the signal recognition particle and its receptor (FtsY). Their homologous GTPase domains interact at the membrane and form a heterodimer in which both GTPases are activated. The prerequisite for protein targeting is the interaction of FtsY with phospholipids. However, the mechanism of FtsY regulation by phospholipids remained unclear. Here we show that the N terminus of FtsY (A domain) is natively unfolded in solution and define the complete membrane-targeting sequence. We show that the membrane-targeting sequence is highly dynamic in solution, independent of nucleotides and directly responds to the density of anionic phospholipids by a random coil-helix transition. This conformational switch is essential for tethering FtsY to membranes and activates the GTPase for its subsequent interaction with the signal recognition particle. Our results underline the dynamics of lipid-protein interactions and their importance in the regulation of protein targeting and translocation across biological membranes.

FIGURE 1.

Crystal structure of FtsY allows definition of complete MTS. A, the domain architecture of the E. coli SRP receptor FtsY. The location of the MTS, the I-box, and the conserved G elements (G1–G5) are indicated. B, superposition of FtsY (blue) and NG+1 (yellow; Protein Data Bank code 2QY9) crystal structures. FtsY and NG+1 are shown in ribbon representation. The MTS present at the N terminus of αN1 is extended in the FtsY structure by two turns (residues 188–195). N and C termini of FtsY are indicated by “N” and “C,” respectively. Two ethylene glycol molecules associated to the hydrophobic face of the MTS are indicated by “EG.” C, N-terminal sequences from various FtsY variants used in this study. The conserved double phenylalanine motif is colored in red.

FIGURE 2.

Analysis of dynamic properties of FtsY in solution by HX-MS. A, relative deuteron incorporation in FtsY for the nucleotide-free (apo), GDP-, and GTP-bound states. Each horizontal block represents an analyzed peptic fragment. Peptides are colored according to the relative deuteron incorporation as indicated. Secondary structure elements and the positions of the MTS, helix αN1, the G elements (G1–G5), and the I-box are marked above the amino acid sequence. B, fast and slow exchanging regions are mapped on the crystal structure shown in a ribbon representation. Segments corresponding to peptic fragments are colored according to the relative deuteron incorporation into the nucleotide-free protein after 10 s and 1, 10, and 30 min in D₂O as indicated.

FIGURE 3.

Anionic phospholipids induce random coil-helix transition of the MTS. Three different MTS peptides were analyzed by CD spectroscopy in the absence of LUVs (white) and the presence of plasma membrane-mimicking LUVs (70% PE and 30% PG; red) and LUVs composed entirely of zwitterionic phospholipids (70% PE and 30% phosphatidylcholine (PC); blue). Samples were treated with 83% trifluoroethanol (TFE) to induce random coil-helix transition (yellow). The MTS of NG+1 (NG+1^pep; MFARLKRSLLKTKENLG) (A), MTS of the NG+1 F196A variant (NG+1 F196A^pep; AARLKRSLLKTKENLG) (C), and MTS of the NG variant (NG^pep; ARLKRSLLKTKENLG) (D) are shown. Only the in vivo functional MTS (NG+1 peptide) undergoes a random coil-helix transition upon interaction with anionic phospholipids. B, secondary structure transition of the three MTS variants is shown as a function of the concentration of anionic phospholipids. The physiologically relevant PG concentration is indicated by a vertical red line. POPG, 1-palmitoyl-2-oleoyl-phosphatidylglycerol. The mean residue ellipticity is shown in degrees cm² dmol⁻¹ residue⁻¹ (deg cm² dmol⁻¹ res⁻¹). E, close-up of the MTS as part of the FtsY structure. The MTS is shown in ribbon representation. The pronounced amphipathic character of the MTS is indicated by a network of bound water molecules at the hydrophilic side and the presence of two ethylene glycol molecules at the hydrophobic side.

FIGURE 4.

Lipid binding properties of FtsY variants. A, the nucleotide dependence of NG+9 and A domain binding to LUVs (PG) was investigated by density gradient flotation analysis. The LUV and pellet fractions are labeled with “L” and “P,” respectively. B and C, binding of NG, NG+1, NG+9, and ΔN1 FtsY to LUVs containing different molar ratios of PG, PE, and cardiolipin (CL). The ΔN1 FtsY lacks the complete MTS sequence and was used as a negative control. D, binding of full-length FtsY and different MTS variants to LUVs (PG). The amount of protein bound to LUVs is given as percentage of total protein. For lipid binding studies, 20 μg (5–10 μm) of proteins and 1.8 mm phospholipids in the absence or presence of 2 mm nucleotides were used. E, ability of FtsY, NG+1 F196A, and FtsY F195/196A to support membrane translocation of the SRP model substrate ppl. The inset shows the SDS-PAGE used to separate ppl and prolactin (pl). The efficiency of ppl translocation was quantified and is given in percent relative to FtsY. The error bars represent the standard deviation between three independent measurements. POPG, 1-palmitoyl-2-oleoyl-phosphatidylglycerol.

FIGURE 5.

MTS is functional in vivo and in vitro. A, fluorescence micrographs of the localization of the MTS of NG+1 (left), NG (middle), and NG+1 F196A (right) fused to the N terminus of GFP. The inset shows the magnification of a representative E. coli cell. The Western blot shows that all MTS-GFP fusions were expressed in E. coli at similar levels after 1 h of induction. B, ability of FtsY, NG+1, NG+2, and NG to support membrane translocation of the SRP model substrate ppl. The inset shows the SDS-PAGE used to separate ppl and prolactin (pl). The efficiency of ppl translocation was quantified and is given in percent relative to FtsY. The error bars represent the standard deviation between three independent measurements. An FtsY variant that contains a functional MTS but is defective in reciprocal GTPase activation in the context of SRP (NG+1 A335W variant) (26) was used as a control and shows a similar translocation defect as observed for NG. C, anionic phospholipids stimulate both the basal GTPase activity of FtsY and its complex assembly with SRP.

FIGURE 6.

Scheme of SRP-mediated protein targeting. FtsY (green) is shown with the A domain (black) and the MTS (red). Prior to membrane interaction, the MTS is dynamic (red line) and undergoes a random coil-helix (red helix) transition upon interaction with anionic phospholipids, which are enriched at the SecYEG translocation channel (Step 1). This conformational switch allows subsequent interaction of FtsY with the RNC-SRP in a GTP-dependent manner (Step 2). Upon SRP-FtsY interaction, the two GTPases present in both proteins are activated (Step 3), and the cargo protein is inserted into the membrane (Step 4). GTP is indicated by T.