Data acquisition and processing statistics.

<p>Data acquisition and processing statistics.</p

Structural transitions and LAMP1 binding of the LASV GP spikes upon acidification.

<p>(A) GP structures at different pHs are shown from side (top row) and top (bottom row). All volumes were filtered to 17-Å resolution, rendered at molecular threshold corresponding to the expected molecular mass, and colored as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005418#ppat.1005418.g001" target="_blank">Fig 1</a>. Residual density corresponding to LAMP1 is colored in green. Inserts in the lower left corners show a close-up of the interface between two spike monomers. Insets in the top right corners show Western blot analysis of GP1 and GP2 subunits. The arrowheads indicate the missing density in the central top part and side of the pH 3 structure.</p

Model for Lassa virus entry.

<p>A schematic representation of a hypothetical entry model, derived from the structures determined in this study, is shown. An approximate range of pH in different cellular compartments is depicted as a color gradient from neutral (blue) to very acidic (red). Lassa virus glycoprotein (GP) spike trimer is depicted in three shades of brown and the two different subunits (GP1 and GP2) are labeled. Lysosome-associated membrane protein 1 (LAMP1), an intracellular Lassa virus receptor, is labeled and colored in green. Viral membrane is colored in blue. Different cellular membranes are labeled and colored in gray. Formation of the crevices between GP1 subunits is indicated with white arrows. See text for full description of the entry model.</p

Comparison of arenavirus and filovirus glycoprotein spike structures.

<p>Structures of Lassa virus spike (LASV, this study), spike of the arenenavirus-like virus infecting snakes (University of Helsinki Virus, UHL; EMD-2424), and Ebola virus spike (EBOV) with (EMD-6003) and without (EMD-6004) the mucin-like domain are shown at the same scale for comparison.</p

Engineering Hydrophobic Protein–Carbohydrate Interactions to Fine-Tune Monoclonal Antibodies

Biologically active conformations of the IgG1 Fc homodimer are maintained by multiple hydrophobic interactions between the protein surface and the N-glycan. The Fc glycan modulates biological effector functions, including antibody-dependent cellular cytotoxicity (ADCC) which is mediated in part through the activatory Fc receptor, FcγRIIIA. Consistent with previous reports, we found that site-directed mutations disrupting the protein–carbohydrate interface (F241A, F243A, V262E, and V264E) increased galactosylation and sialylation of the Fc and, concomitantly, reduced the affinity for FcγRIIIA. We rationalized this effect by crystallographic analysis of the IgG1 Fc F241A mutant, determined here to a resolution of 1.9 Å, which revealed localized destabilization of this glycan–protein interface. Given that sialylation of Fc glycans decreases ADCC, one explanation for the effect of these mutants on FcγRIIIA binding is their increased sialylation. However, a glycan-engineered IgG1 with hypergalactosylated and hypersialylated glycans exhibited unchanged binding affinity to FcγRIIIA. Moreover, when we expressed these mutants as a chemically uniform (Man₅GlcNAc₂) glycoform, the individual effect of each mutation on FcγRIIIA affinity was preserved. This effect was broadly recapitulated for other Fc receptors (FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIB). These data indicate that destabilization of the glycan–protein interactions, rather than increased galactosylation and sialylation, modifies the Fc conformation(s) relevant for FcγR binding. Engineering of the protein–carbohydrate interface thus provides an independent parameter in the engineering of Fc effector functions and a route to the synthesis of new classes of Fc domain with novel combinations of affinities for activatory and inhibitory Fc receptors

Chemical and Structural Analysis of an Antibody Folding Intermediate Trapped during Glycan Biosynthesis

Human IgG Fc glycosylation modulates immunological effector functions such as antibody-dependent cellular cytotoxicity and phagocytosis. Engineering of Fc glycans therefore enables fine-tuning of the therapeutic properties of monoclonal antibodies. The N-linked glycans of Fc are typically complex-type, forming a network of noncovalent interactions along the protein surface of the Cγ2 domain. Here, we manipulate the mammalian glycan-processing pathway to trap IgG1 Fc at sequential stages of maturation, from oligomannose- to hybrid- to complex-type glycans, and show that the Fc is structurally stabilized following the transition of glycans from their hybrid- to complex-type state. X-ray crystallographic analysis of this hybrid-type intermediate reveals that N-linked glycans undergo conformational changes upon maturation, including a flip within the trimannosyl core. Our crystal structure of this intermediate reveals a molecular basis for antibody biogenesis and provides a template for the structure-guided engineering of the protein–glycan interface of therapeutic antibodies

Chemical and Structural Analysis of an Antibody Folding Intermediate Trapped during Glycan Biosynthesis

Human IgG Fc glycosylation modulates immunological effector functions such as antibody-dependent cellular cytotoxicity and phagocytosis. Engineering of Fc glycans therefore enables fine-tuning of the therapeutic properties of monoclonal antibodies. The N-linked glycans of Fc are typically complex-type, forming a network of noncovalent interactions along the protein surface of the Cγ2 domain. Here, we manipulate the mammalian glycan-processing pathway to trap IgG1 Fc at sequential stages of maturation, from oligomannose- to hybrid- to complex-type glycans, and show that the Fc is structurally stabilized following the transition of glycans from their hybrid- to complex-type state. X-ray crystallographic analysis of this hybrid-type intermediate reveals that N-linked glycans undergo conformational changes upon maturation, including a flip within the trimannosyl core. Our crystal structure of this intermediate reveals a molecular basis for antibody biogenesis and provides a template for the structure-guided engineering of the protein–glycan interface of therapeutic antibodies

Estimated time of most recent common ancestor (TMRCA) for Yamagata-lineage clade 2 and clade 3 viruses.

<p>Estimated time of most recent common ancestor (TMRCA) for Yamagata-lineage clade 2 and clade 3 viruses.</p

Estimated emergence of nonsynonymous substitutions along the major trunk lineage of the gene phylogenies of Victoria- and Yamagata-lineage virus.

<p>Substitutions are summarized from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006749#ppat.1006749.s001" target="_blank">S1 Fig</a> for (A) Victoria-lineage, (B) Yamagata-lineage, (C) Yamagata-lineage clade 2 (B/Massachusetts/02/2012 clade), and (D) Yamagata-lineage clade 3 (B/Wisconsin/1/2010 clade), with only substitutions emerging after 1995 shown for clarity. Circles represent median and lines represent 95% HPD estimates of time of emergence across 1,000 posterior trees.</p

Maximum clade credibility tree inferred from 1,169 Yamagata-lineage HA gene sequences and corresponding genotype constellations.

<p>Branches of the phylogeny are labeled with amino acid substitutions occurring along the phylogenetic ‘trunk’ and colored by well-supported clade distinction (see legend). Clade classifications of each gene are similarly indicated by colored bars. White bars indicate that no sequence was available for that gene. Nodes with greater than 0.70 posterior probability support are shown with circle node shapes.</p