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. 2017 Oct 1;73(Pt 10):822-828.
doi: 10.1107/S2059798317013353. Epub 2017 Sep 29.

X-ray and EM structures of a natively glycosylated HIV-1 envelope trimer

Harry B Gristick  1 , Haoqing Wang  1 , Pamela J Bjorkman  1
Affiliations

X-ray and EM structures of a natively glycosylated HIV-1 envelope trimer

Harry B Gristick et al. Acta Crystallogr D Struct Biol. .

Abstract

The structural and biochemical characterization of broadly neutralizing anti-HIV-1 antibodies (bNAbs) has been essential in guiding the design of potential vaccines to prevent infection by HIV-1. While these studies have revealed critical mechanisms by which bNAbs recognize and/or accommodate N-glycans on the trimeric envelope glycoprotein (Env), they have been limited to the visualization of high-mannose glycan forms only, since heterogeneity introduced from the presence of complex glycans makes it difficult to obtain high-resolution structures. 3.5 and 3.9 Å resolution crystal structures of the HIV-1 Env trimer with fully processed and native glycosylation were solved, revealing a glycan shield of high-mannose and complex-type N-glycans that were used to define the complete epitopes of two bNAbs. Here, the refinement of the N-glycans in the crystal structures is discussed and comparisons are made with glycan densities in glycosylated Env structures derived by single-particle cryo-electron microscopy.

Keywords: HIV-1 envelope; N-linked glycans; X-ray crystallography; single-particle cryo-EM.

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Figures

Figure 1
Figure 1
High-mannose N-glycans are added to Asn-X-Ser/Thr sequons in the endoplasmic reticulum (first two panels) and can be modified into complex-type glycans in the Golgi apparatus (third and fourth panels). Inhibitors such as kifunensine or growth in mutant mammalian cell lines (GnTI− expression) limits N-linked glycan addition to high-mannose forms. Complex-type glycans are distinguished from high-mannose glycans by the addition of a core fucose at the base and negatively charged sialic acids at the tips.
Figure 2
Figure 2
Side and top views of PDB entry 5t3x: 3.9 Å resolution crystal structure of natively glycosylated Env trimer (gp120, brown; gp41, pink) in complex with bNAb Fabs IOMA (blue) and 10-1074 (green). PNGSs modeled as high-mannose (cyan) or complex glycans (magenta) are shown as spheres.
Figure 3
Figure 3
Cremer–Pople analysis of the initial model (left) and the final model (PDB entry 5t3x) after iterative building and refinement (right). Note that a larger fraction of the glycans in the initial model were in high-energy conformations with significant puckering, which shifted to the more common lower energy conformations following refinement.
Figure 4
Figure 4
Comparison of the initial 2F o (left), final 2F o (center) and OMIT (right) maps for the 3.9 and 3.5 Å resolution structures at PNGSs N156gp120 (first row), N276gp120 (second row), N332gp120 (third row) and N386gp120 (fourth row). All maps are contoured at 0.8σ.
Figure 5
Figure 5
Comparison of the density at selected PNGSs from the 6.2 and 8.9 Å resolution cryo-EM maps.
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