Purification and crystallization reveal two types of interactions of the fusion protein homotrimer of Semliki Forest virus

Abstract

The fusion proteins of the alphaviruses and flaviviruses have a similar native structure and convert to a highly stable homotrimer conformation during the fusion of the viral and target membranes. The properties of the alpha- and flavivirus fusion proteins distinguish them from the class I viral fusion proteins, such as influenza virus hemagglutinin, and establish them as the first members of the class II fusion proteins. Understanding how this new class carries out membrane fusion will require analysis of the structural basis for both the interaction of the protein subunits within the homotrimer and their interaction with the viral and target membranes. To this end we report a purification method for the E1 ectodomain homotrimer from the alphavirus Semliki Forest virus. The purified protein is trimeric, detergent soluble, retains the characteristic stability of the starting homotrimer, and is free of lipid and other contaminants. In contrast to the postfusion structures that have been determined for the class I proteins, the E1 homotrimer contains the fusion peptide region responsible for interaction with target membranes. This E1 trimer preparation is an excellent candidate for structural studies of the class II viral fusion proteins, and we report conditions that generate three-dimensional crystals suitable for analysis by X-ray diffraction. Determination of the structure will provide our first high-resolution views of both the low-pH-induced trimeric conformation and the target membrane-interacting region of the alphavirus fusion protein.

FIG. 1.

Sucrose gradient flotation analysis of ectodomains and liposomes. (A) Purified ectodomains were mixed with liposomes containing [³H]cholesterol as a tracer, treated at pH 5.5 for 10 min at 37°C, returned to neutral pH, and analyzed without detergent treatment. The sample was loaded in the bottom of a sucrose step gradient, centrifuged in a TLS55 rotor at 55,000 rpm for 3 h at 4°C, and fractionated into seven 300-μl fractions. The fractions were precipitated by acid, solubilized in sample buffer at 95°C, reduced and alkylated, and analyzed by SDS-PAGE and Coomassie blue staining. An aliquot of the starting ectodomain prep is shown (ectos). Fraction 1 is the top of the gradient. The positions of E1*, E2*, p62*, and BSA are indicated. (B) Samples were analyzed by flotation as done for panel A, either without detergent pretreatment (open squares) or after treatment for 1 h at room temperature with the indicated detergent at a final concentration of 1.5%. The sucrose gradients also contained the indicated detergent except for the OG sample marked ± (filled diamonds). The amount of [³H]cholesterol in aliquots of each fraction was measured by liquid scintillation counting. CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate.

FIG. 2.

Sucrose sedimentation gradient purification of the E1* homotrimer. Liposome-bound homotrimer was separated by flotation in the absence of detergent as done for Fig. 1A, and a sample (∼100 μg of protein) was solubilized in 1.5% OG for 1 h at room temperature and was layered on top of a 7 to 15% sucrose gradient containing 1% OG. The gradient was centrifuged for 22 h at 35,000 rpm in an SW41 rotor, fractionated from the bottom, and assayed by SDS-PAGE and silver staining after solubilization in SDS-sample buffer at 30°C to preserve the homotrimer (A). The [³H]cholesterol marker was assayed by liquid scintillation counting (B). Fractions were pooled as indicated beneath the gel depicted in panel A, lipid extracts were prepared, and lipid phosphate was quantitated as outlined in Materials and Methods. Background in this assay was ∼3 nmol of P_i.

FIG. 3.

Analysis of microconcentrator-purified E1* homotrimer. (A) Samples of the starting ectodomains (lane 1, Ectos) or the final microconcentrator-purified homotrimer (Purified HT) were incubated in SDS-containing sample buffer for 3 min at 30°C or were reduced at 95°C and alkylated before analysis by SDS-PAGE and Coomassie blue staining. The last three lanes show increasing amounts of the purified material (10, 20, and 30 μl). The positions of the homotrimer (HT) and the monomeric proteins E1*, E2*, p62*, and BSA are indicated. (B) Samples of the concentrator-purified homotrimer were incubated for 1 h at 37°C with 0.5% TX-100 and 125 μg of trypsin/ml (lanes 1 and 2). For comparison, samples of the stock trypsin (lane 3) and of the purified E1*HT (lane 4) are shown. The samples were solubilized in SDS-sample buffer at 30°C to retain the HT (lane 1) or at 95°C with reduction and alkylation (lanes 2 to 4) and were analyzed by SDS-PAGE and Coomassie blue staining. The two arrows indicate the positions of reduced and nonreduced trypsin.

FIG. 4.

Dynamic light-scattering analysis of the purified E1* homotrimer. Each panel shows a single experiment containing the cumulative data from 50 to 100 measurements graphed as intensity versus the R_h (in nanometers on a log scale). The data are not corrected for the changes in solution viscosity due to detergent and salt differences between samples; therefore, the R_h values shown are intended only for relative qualitative comparisons. (A) Analysis of purified E1* homotrimer (∼0.5 mg/ml) in buffer containing 1 M NaCl, 50 mM Tris-HCl, pH 7.5, and the detergent OG at (0.7%) (A1) or below (0.08%) (A2) the CMC. (B) Analysis of purified E1* homotrimer in buffer containing 0.7% OG, 50 mM Tris-HCl, pH 7.5, and 100 mM NaCl (B1). Panel B2 gives the analysis of the sample from panel B1 after addition of NaCl to a final concentration of 1 M and incubation for 5 min.

FIG. 5.

3D crystals of the E1* homotrimer grown at 19°C. (A) An example of the rod-shaped crystals grown by hanging-drop vapor diffusion in 11% PEG 400, 3.0 mM LDAO, 0.25 M NaCl, 25 mM Tris-HCl, pH 7.5, and 2.5 mg of protein/ml (initial concentrations in the drop). (B) Hexagonal plate-shaped crystals grown in 7.5% PEG 1000, 1.5 mM LDAO, 0.25 M NaCl, 25 mM Tris-HCl, pH 7.5, and 2.5 mg of protein/ml. Some precipitate is also seen. (C) Rounded plate shaped-crystals grown in 5% PEG 1000, 3.0 mM LDAO, 0.25 M NaCl, 25 mM Tris-HCl, pH 7.5, and 2.5 mg of protein/ml.

FIG. 6.

3D crystals and X-ray diffraction of the E1* homotrimer. The left panel shows a crystal grown from 6% PEG 400, 0.25 M NaBr, 10 mM HoCl₃, 25 mM Tris-HCl, pH 7.5, and 15 mM DDAO, by use of the hanging-drop vapor diffusion technique as explained in Materials and Methods. The crystals are hexagonal prisms that grow to approximately 200 μm in length and to approximately 80 μm in thickness. The right panel shows the diffraction pattern from a similar crystal, a 0.2° oscillation image taken at beam-line X06SA at the Swiss Light Source in Viligen, Switzerland. The detector was a charge-coupled device made by MAR Research (Hamburg, Germany). The wavelength of the X-ray beam was 1.1808 Å, the crystal-to-detector distance was 200 mm, and the exposure time was 2 s. The resolution at the edge of the detector was 3.18 Å.