Received 20 June 2001
Crystallization and preliminary diffraction studies of the ectodomain of the envelope glycoprotein D from herpes simplex virus 1 alone and in complex with the ectodomain of the human receptor HveA
Andrea Carfí,a+ Haiyun Gong,a++ Huan Lou,b Sharon H. Willis,b Gary H. Cohen,b Roselyn J. Eisenbergc and Don C. Wileya,d*
aChildren's Hospital, Department of Medicine and Howard Hughes Medical Institute, 320 Longwood Avenue, Boston, MA 02115, USA,bDepartment of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA,cDepartment of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA, and dDepartment of Molecular and Cellular Biology, Howard Hughes Medical Institute, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA
Gycoprotein D (gD) is a glycoprotein expressed on the surface of several human and animal alpha herpes viruses. Binding of gD to cell-surface receptors has been shown to be necessary for herpes simplex virus 1 and 2 (HSV-1 and HSV-2) cell entry. The gD ectodomain consists of 316 residues and has no sequence homology to any other proteins of known structure. Two fragments of the HSV-1 gD ectodomain (gD22-260: residues 22-260 and gD285: residues 1-285) have been crystallized in two crystal forms. The complex between gD285 and the ectodomain of HveA, a gD cellular receptor member of the tumor necrosis factor (TNFR) superfamily, has also been crystallized. Moreover, insect-cell-expressed selenomethionine-substituted gD285 has been purified and crystallized alone and in complex with HveA.
Alpha herpes viruses are large double-stranded DNA viruses that infect humans [herpes simplex virus 1 and 2 (HSV-1 and HSV-2)] and animals [e.g. bovine herpes virus 1 (BHV-1) and porcine pseudorabies virus (PrV)]. HSV-1 and HSV-2 are widespread in the human population: HSV-1 is normally associated with oral and ocular lesions, whereas HSV-2 is isolated primarily from genital and oral lesions. After an initial infection in epithelial cells, the virus spreads to host neuronal cells where it can remain latent for life (Roizman & Sears, 1987). The mechanism of infection of this family of viruses is complex and not fully understood. At least five glycoproteins (gB, gC, gD and the gH/gL complex) on the virion envelope are involved in HSV cell entry (reviewed in Spear et al., 2000). Among those, glycoprotein D (gD) has been shown to mediate binding to cellular receptors (reviewed in Campadelli-Fiume et al., 2000; Spear et al., 2000).
HSV-1 gD is a 369-residue glycoprotein with an N-terminal ectodomain of 316 residues and three N-linked oligosaccharide-attachment sites (Watson et al., 1982). The protein has six cysteine residues which form three disulfide bonds (Long et al., 1992; HSV-1 and HSV-2 gD ectodomains have 85% sequence identity) and five methionines. In the past three years, several gD receptors have been identified (Campadelli-Fiume et al., 2000; Spear et al., 2000). The first receptor to be cloned was HveA, a member of the TNFR/NGFR superfamily expressed on the surface of activated T-lymphocytes, where it mediates HSV-1 and HSV-2 entry (Montgomery et al., 1996).
HveA (also called HVEM or TNFRSF14) is a type I membrane protein with an ectodomain of 162 residues and two N-linked oligosaccharide-attachment sites. Sequence alignments show that the first N-terminal 100 residues have a significant sequence identity (close to 30%) with the TNFR family member TNFR-1, whose structure is known (Banner et al., 1993; Naismith et al., 1996).
gD is an important target for antiviral agents and soluble forms of gD are in clinical trials as vaccines against HSV-1 and HSV-2. To gain insights into the role of gD in HSV entry, we have started structural studies on gD (Krummenacher et al., 1998; Nicola et al., 1997; Rux et al., 1998; Whitbeck et al., 1997; Willis et al., 1998) and on complexes of gD with cellular receptors. Here, we report the crystallization and preliminary diffraction data of a recombinant gD ectodomain alone and in complex with the ectodomain of HveA.
Both the HveA ectodomain (residues 1-162 plus a C-terminal His5 tag) and two soluble C-terminally truncated forms of gD (residues 1-306 and 1-285) were purified from Sf9 insect-cell culture supernatants (Sisk et al., 1994). HveA was purified on an Ni2+-resin followed by size-exclusion chromatography on a Pharmacia analytical Superdex-200 column. gD285 and gD306 were purified using DL6-antibody affinity chromatography with pH 11 elution (DL-6 recognizes a linear epitope, residues 272-279, near the C- terminus of gD; Isola et al., 1989) followed by ion exchange on a MonoQ column (Pharmacia) and size-exclusion chromatography on an analytical Superdex-200 column (Pharmacia). Selenomethionine-substituted gD285 was produced following published protocols (for a review, see Bellizzi et al., 1999) that were slightly modified. Sf9 cells were grown to a density of 106 cells ml-1 in 1 l spinner flasks in methionine-containing Sf9-II00 media (GIBCO) (500 ml). 24 h post-infection, the cells were gently spun down and moved to methionine-free Grace's media (GIBCO) to remove the intracellular pull of methionine. 12-14 h later the cells were transferred to a fresh methionine-free Grace's media supplemented with 5% dialyzed fetal bovine serum (FBS; Sigma) and 75 mg ml-1 selenomethione (Sigma) for an additional 48 h. The protein was then purified from the Sf9 insect-cell culture supernatants by the same methods as the native protein. The yield for the selenomethionine protein was approximately 50-60% compared with that normally achieved in a native protein preparation. Amino-acid analysis showed that 85% selenomethionine incorporation had been obtained.
The gD285-HveA complex was prepared by titration of gD285 with increasing amounts of HveA, observing the gel shift with native PAGE (without SDS or -mercaptoethanol). The complex was then purified by size-exclusion chromatography on an analytical Superdex-200 column, which allowed separation of the complex from the excess unbound gD.
Preliminary crystallization trials were performed by the hanging-drop vapour-diffusion method. Typically, 1 µl of protein solution (5.5-12 mg ml-1 in 10 mM Tris-HCl pH 7.0 and 100 mM NaCl) was mixed with an equal volume of well solution. Once crystals were obtained, crystal growth was optimized using the sitting-drop method with larger protein droplets.
We first tried to crystallize gD306. Small crystals (approximately 0.15-0.2 mm per side; Fig. 1b) grow in about three months in droplets equilibrated with solutions containing 50 mM MES pH 6.0 as buffer and 1.6 M ammonium sulfate as precipitating agent. SDS gels of washed crystals showed that the protein had been partially proteolysed during the long crystallization period. N-terminal sequencing showed that the crystals contained a gD core starting at residue 21 of the mature protein and this information together with MALDI mass spectrometry indicates that the new C-terminus is in the proximity of residue 260.
| || Figure 1 |
Crystals of (a) gD285, (b) gD22-260 and (c) gD285-HveA
Crystals of gD285 or Se-Met gD285 were obtained in the pH range 6.0-9.0 using 1.4 M ammonium sulfate as precipitating agent. The largest crystals (0.6 × 0.3 × 0.3 mm) (Fig. 1a) were grown by mixing 3 µl of purified complex solution with an equal volume of a reservoir solution containing 50 mM Bicine pH 9.0 (or 50 mM MES pH 6.0) as buffer and 1.6 M ammonium sulfate as precipitating agent in sitting-drop experiments. SDS gels and N-terminal sequencing on washed crystals confirmed the presence of the full protein (residues 1-285) in the crystals.
More recently, crystals of the gD285-HveA and SeMet gD285-HveA complexes have been obtained (Fig. 1c). The complex was prepared by titrating gD285 with increasing amounts of HveA162 and observing the gel shift with native PAGE and was further purified by size-exclusion chromatography on a Superdex-200 column. Large crystals (0.8 × 0.3 × 0.2 mm) were obtained in sitting-drop experiments by mixing 3 µl of purified complex solution with an equal volume of a reservoir solution containing 50 mM Tris-HCl pH 8.0 as buffer and 1.8 M ammonium sulfate and 5% PEG 400 as precipitating agents.
For X-ray data collection at cryogenic temperature, crystals were transferred stepwise to cryoprotectant solutions containing 20% glycerol (gD285 and gD22-260) or 20% ethylene glycol (gD285-HveA) in crystallization buffer and flash-cooled at 100 K in N2 gas. Preliminary characterization of the crystals was carried out in the laboratory on a MAR 30 cm detector mounted on a GX-13 X-ray generator with Charles Supper double focusing mirrors. Owing to the small size of gD22-260 crystals and to the weak diffraction proprieties of the gD285 and gD285-HveA crystals, synchrotron X-ray sources had to be used for data collection (Table 1). The HKL suite was used for data integration and scaling (Otwinoski & Minor, 1997). The CCP4 suite was used for further data processing and analysis (Collaborative Computational Project, Number 4, 1994).
A 2.80 Å data set from gD22-260 crystals was collected at the BIOCARS 14BMC beamline at the APS on a Q4-CCD detector and at 1.00 Å wavelength. The crystals belong to the space group P42212 (Table 1), with unit-cell parameters a = b = 158.67, c = 155.70 Å and at least four molecules in the asymmetric unit (VM = 3.8, corresponding to 67% solvent).
In the case of gD285, several crystals were screened. A 2.85 Å native data set was collected at the X25 beamline at BNL-NSLS on a Brandeis B4-CCD detector at 0.9754 Å wavelength. The data were indexed and integrated in the tetragonal space group P422, with unit-cell parameters a = b = 131.43, c = 83.27 Å (Table 1). The diffraction images were characterized by the presence of streaked reflections in the b* and a* directions and also by the absence of many predicted reflections. A native Patterson map contained a peak with half the intensity of the origin peak at u = 0.5, v = 0.5 and w = 0.4 (Fig. 2), which indicates the presence of two molecules in the asymmetric unit (VM = 2.75 Å3 Da-1) related by translational non-crystallographic symmetry. The presence of streaked reflections in the diffraction pattern might be indicative of static disorder in the crystals.
| || Figure 2 |
w = 0.4 section in the gD285 native Patterson map (0 < u < 1 and 0 < v < 1). The peak at u = 0.5, v = 0.5 in this section has half the intensity of the origin peak.
For the gD285-HveA crystals, a 2.65 Å resolution native data has been collected at the A1 beamline at CHESS on a Q4 CCD detector (Table 1). The data were indexed and integrated in the P3121 space group, with unit-cell parameters a = b = 129.15, c = 80.94 Å, and presumably contain one complex molecule per asymmetric unit (VM = 3.7 Å3 Da-1, corresponding to 67% solvent if we assume a 1:1 complex).
This work was supported in part by NIH grants RO1 NS 36731, RO1 NS 30603 and RO1 AI 18298 to GHC and RJE. AC was supported by a fellowship from the Irvington Institute for Immunological Research. DCW is an investigator of the HHMI.
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