Crystallization and preliminary crystallographic analysis of the measles virus hemagglutinin in complex with the CD46 receptor

Abstract

The measles virus (MV) hemagglutinin (MV-H) mediates the attachment of MV particles to cell-surface receptors for entry into host cells. MV uses two receptors for attachment to host cells: the complement-control protein CD46 and the signalling lymphocyte activation molecule (SLAM). The MV-H glycoprotein from an Edmonston MV variant and the MV-binding fragment of the CD46 receptor were overproduced in mammalian cells and used to crystallize an MV-H-CD46 complex. Well diffracting crystals containing two complexes in the asymmetric unit were obtained and the structure of the complex was solved by the molecular-replacement method.

Figure 1

Crystals of the isolated MV-H fragment and the MV-H–CD46 complex. The top row shows crystals prepared with 2 M ammonium sulfate, 5% MPD and 0.1 M sodium cadodylate pH 6.5. The bottom row shows crystals prepared with 12% PEG 8000, 0.2 M ammonium sulfate, 2% PEG 400 and 0.1 M sodium MES pH 6.5. (a) Crystals of the MV-H fragment. (b) Crystals of endoglycosidase H-treated MV-H fragment. (c, d) Crystals of the MV-H–CD46 complex. (e) Crystals of the complex prepared with the addition of 1% 1,2,3-heptanetriol to the PEG 8000 crystallization condition. (f) Crystals of the complex prepared with the addition of 1% 1,2,3-heptanetriol and 1 mM sodium tungstate (Na₂WO₄).

Figure 2

Identification of the proteins in the MV-H–CD46 crystals. MALDI–TOF analysis of redissolved crystals. The spectrum shows two peaks that correspond to proteins with molecular masses of 55 024 and 16 916 Da, corresponding to MV-H and CD46, respectively. Mass determination was calculated from the single charged ion of the proteins. The inset shows 12% SDS–PAGE analysis under reducing conditions of redissolved crystals (lane 2) and molecular mass markers (lane 1). The mass (kDa) and migration of the marker proteins are indicated.

Figure 3

X-ray diffraction image from a native MV-H–CD46 crystal (Na7 in Table 1 ▶). Rings are shown at the detector edge and at three additional resolution limits.