Structure, binding, and activity of Syd, a SecY-interacting protein

Abstract

The Syd protein has been implicated in the Sec-dependent transport of polypeptides across the bacterial inner membrane. Using Nanodiscs, we here provide direct evidence that Syd binds the SecY complex, and we demonstrate that interaction involves the two electropositive and cytosolic loops of the SecY subunit. We solve the crystal structure of Syd and together with cysteine cross-link analysis, we show that a conserved concave and electronegative groove constitutes the SecY-binding site. At the membrane, Syd decreases the activity of the translocon containing loosely associated SecY-SecE subunits, whereas in detergent solution Syd disrupts the SecYEG heterotrimeric associations. These results support the role of Syd in proofreading the SecY complex biogenesis and point to the electrostatic nature of the Sec channel interaction with its cytosolic partners.

FIGURE 1.

Binding of Syd onto the SecYEG-Nanodisc. A, indicated amount of Syd was incubated with 3 μg of Nd-SecYEG in TSG buffer (5 min at room temperature). The samples were analyzed by native-PAGE followed by Coomassie Blue staining of the gel. The molecular weight markers (MWM) are ferritin (440 kDa), catalase (232 kDa), and bovine serum albumin (66/132 kDa). The Nd-SecYEG preparation contains some free membrane scaffold protein (MSP) dimers that migrate near the 66-kDa marker. B, ¹²⁵I-Syd (∼25,000 cpm; 150 ng) was incubated with Nd-SecYEG, either wild-type or carrying the indicated deletion in the SecY subunit. Samples were analyzed by native-PAGE and phosphorimaging. C, ¹²⁵I-Syd (∼250,000 cpm; 1.25 μg) was analyzed by sucrose density centrifugation with the SecYEG-Nanodiscs (∼35 μg), either wild-type (WT) or carrying the indicated deletion in the SecY subunit. The gradient (6–13%) was prepared in 50 mm Tris, pH 7.9, 5% glycerol, 1 mm DTT containing 100 mm NaCl, or no salt as indicated. The centrifugation was at 197,000 × g for 16 h at 4 °C in a Beckman SW41 rotor. Equal fractions were collected and analyzed by SDS-PAGE and phosphorimaging.

FIGURE 2.

Isolation of the Syd-SecYEG-Nanodisc complex and mass determination. A, SecYEG-Nanodisc (∼200 μg) incubated with a molar excess of Syd (∼100 μg) and then applied onto a Superdex 200 HR10/10 column equilibrated in TSG buffer. The fractions containing the complex Nd-SecYEG-Syd were pooled and concentrated to 1.2 mg/ml for subsequent multiangle light scattering analysis. B, multiangle light scattering of Syd, Nd-SecYEG, and Nd-SecYEG-Syd. In each case, about 100 μg of protein is loaded onto a Superdex 200 HR 10/30 column equilibrated in 10 mm HEPES, 50 mm NaCl, pH 7.4.

FIGURE 3.

Atomic structure and surface electrostatic of Syd and SecYEG. A, ribbon diagram representation of Syd (β-sheets, yellow; α-helices, magenta; loops, green) and space-filling representation showing the concave structure of the protein (180° rotation compared with right panel). Arrows denote the position of the “stalk” regions that consist of negatively charged protruding loops that delineate the concave region. B, electrostatic potential of the concave (left) and convex surface (right, 180° rotation). Blue and red represent electropositive and electronegative potential, respectively. The surface potential was set between -2.5 and +2.5 kT/e using the solvent-accessible area option of the software. C, electrostatic map of the archaeal SecY complex (Protein Data Bank code 1RH5) showing the position of the positively charged loops C4 and C5. The surface potential was generated using the parameters described in B. Positive charges are located near the predicted location of the phospholipids head group. Note that in E. coli, the loops C4 and C5 contain additional basic residues compared with Methanococcus jannaschii. In particular, three arginine residues are located at the tip of the loop C4 in the E. coli complex.

FIGURE 4.

Contact surface between Syd and SecY and exclusion of SecA. A, location of the unique cysteine residues (90, 97, 115, and 135) introduced at the surface of Syd is indicated in yellow. B, purified Syd proteins (each 2 μg) were mixed with IMVs (5 μg) enriched for the SecY complex, either wild-type (WT) or carrying the cysteine mutation at position 255 (SecY255C) in 100 μl of TSG buffer (without DTT). After 5 min of incubation at room temperature, the disulfide bond formation was stopped with N-ethylmaleimide (NEM; 10 mm). Samples were analyzed by SDS-PAGE followed by immunostaining with a polyclonal antibody directed against SecY. The presence of N-ethylmaleimide during the incubation prevents the formation of the SecY-Syd cross-link (lower panel, left lane). C, IMVs SecY255C (5 μg) were incubated with a fixed amount of Syd-115C (2 μg) and a variable amount of SecA (0.0–5.0 μg) in 100 μl of TSG buffer without DTT. Samples were analyzed by SDS-PAGE followed by immunostaining with anti-SecY antibodies. D, about 3 μg of SecYEG-Nanodisc was incubated with a fixed amount of SecA (1 μg) and a variable amount of Syd (0.0–1.0 μg) in 50 μl of TSG buffer. After 5 min of incubation at room temperature, samples were analyzed by native-PAGE and Coomassie Blue staining of the gel.

FIGURE 5.

Translocation activity of the SecY complex in the presence of Syd. A, SecY complex, wild-type (WT) or carrying the mutation secY24(G240D) or SecEΔ7-67, was overproduced in E. coli, and the IMVs were immunostained with anti-SecY antibodies. The SecY mutant complex cannot be overproduced to the same level as the wild type, probably due to a higher instability. B, IMVs were urea-stripped, and their concentration was adjusted so that a comparable amount of SecY complex is tested in the corresponding in vitro translocation assays. The IMVs were premixed with the indicated amount of purified Syd before addition of the translocation substrate ¹²⁵I-proOmpA and SecA (0.25 μg). Translocation was initiated with ATP (1 mm) for 10 min at 37 °C. After proteinase K digestion (10 min on ice), samples were analyzed by SDS-PAGE and phosphorimaging. The results were quantified by densitometry and expressed on the graph curve (right panel).

FIGURE 6.

Stability of the SecY complex in the presence of Syd. A, ¹²⁵I-SecY complex (∼75,000 cpm; 0.75 μg) was incubated with the indicated amount of purified Syd protein. The incubation was for 10 min at 22 °C in TSG buffer containing the indicated amount of detergent. Samples were analyzed by blue-native PAGE and phosphorimaging. B, detergent-soluble SecYEG complex (600 μg in 50 mm Tris, pH 7.9, 600 mm NaCl, 5% glycerol, 0.03% DDM) was mixed with a molar excess of Syd (450 μg in TSG buffer) and incubated for 5 min with 0.03% DDM at room temperature or with 0.1% Triton X-100 (TX-100) at 37 °C. The mixtures were applied onto a Superdex 200 HR10/30 gel filtration column equilibrated in 50 mm Tris, pH 7.9, 300 mm NaCl, 5% glycerol containing either 0.03% DDM or 0.1% Triton X-100 as indicated. The eluted fractions were analyzed by SDS-PAGE and Coomassie staining.