Visualization of membrane protein domains by cryo-electron microscopy of dengue virus - PubMed (original) (raw)

. 2003 Nov;10(11):907-12.

doi: 10.1038/nsb990. Epub 2003 Oct 5.

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Visualization of membrane protein domains by cryo-electron microscopy of dengue virus

Wei Zhang et al. Nat Struct Biol. 2003 Nov.

Abstract

Improved technology for reconstructing cryo-electron microscopy (cryo-EM) images has now made it possible to determine secondary structural features of membrane proteins in enveloped viruses. The structure of mature dengue virus particles was determined to a resolution of 9.5 A by cryo-EM and image reconstruction techniques, establishing the secondary structural disposition of the 180 envelope (E) and 180 membrane (M) proteins in the lipid envelope. The alpha-helical 'stem' regions of the E molecules, as well as part of the N-terminal section of the M proteins, are buried in the outer leaflet of the viral membrane. The 'anchor' regions of E and the M proteins each form antiparallel E-E and M-M transmembrane alpha-helices, leaving their C termini on the exterior of the viral membrane, consistent with the predicted topology of the unprocessed polyprotein. This is one of only a few determinations of the disposition of transmembrane proteins in situ and shows that the nucleocapsid core and envelope proteins do not have a direct interaction in the mature virus.

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Figures

Figure 1

Figure 1

The dengue virus structure. (a) Stereo view of the viral surface at a resolution of 12.0 Å. The brown triangle demarcates the limits of one icosahedral asymmetric unit as defined by the five- and three-fold axes. Note the two protrusions per monomer corresponding to the glycosylation sites at Asn67 (yellow) and Asn153 (red). (b) A central cross section looking down an icosahedral three-fold axis, showing the polygonal shape of the membrane. The darkness of the shading is proportional to the magnitude of the cryo-EM density. Viral components are labeled. Maximum density heights are plotted below on a relative scale as a function of radius. (c) A radial cryo-EM density section at a radius of 185 Å, corresponding to the center of the lipid membrane, highlighting the herringbone arrangement of the three E dimers. The density is indicated in gray scale, with the highest density being the blackest. Shown also in brown is the limit of one icosahedral asymmetric unit. The boundaries of the E glycoprotein dimers are also indicated. The E dimer on the icosahedral twofold axis is red, whereas the monomers of the general-position dimer are blue and green. The transmembrane helices are viewed in cross section and marked for the green monomer according to the nomenclature of Figure 2.

Figure 2

Figure 2

Secondary structural predictions based on the primary sequences of the E and M stem-anchor regions. Helical coils represent the E stem (E-H1, E-H2) and transmembrane anchor (E-T1, E-T2) and the M stem (M-H) and transmembrane anchor (M-T1, M-T2) regions.

Figure 3

Figure 3

Stereoscopic diagrams showing the fit of the Cα backbones for the E and M regions into the cryo-EM density (gray) of the outer lipid (green) leaflet associated with the E dimer on the icosahedral two-fold axis. E ectodomains I, II and III are red, yellow and blue, respectively; stem-anchor region of E, cyan; M protein, orange; cryoEM density of the lipid bilayers, green. The stem and transmembrane helices are labeled with the nomenclature shown in Figure 2. Contour levels are chosen arbitrarily. The contour level for the lipid (green) is lower than that for the protein (gray). (a) Side view showing E and M monomers. (b) Enlarged view of a with a +50° rotation about the vertical axis to more clearly show the fit of E-H1 into the density. (c) Enlarged view of a with a –20° rotation about the vertical axis to more clearly show the fit of E-H2 and M-H into the density. (d) Top view of helices E-H1, E-H2 and M-H.

Figure 4

Figure 4

Diagrams of the dengue virus ectodomain and transmembrane domain proteins. The volume occupied by the ectodomain of an E monomer is pink (domain I), yellow (domain II) and lilac (domain III). The stem and anchor helices of E and M are blue and orange, respectively. Helices are identified by the nomenclature shown in Figure 2. CS represents the conserved sequence between E-H1 and E-H2. (a) View as in Figure 3a. (b) View as in Figure 3d with the superimposed E ectodomain homodimer.

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