Biology subjects

Berndsen, Z. T.

Publications and source records attributed to Berndsen, Z. T..

3 recordsLinked to original sources

bioRxiv2019

A Network-based approach for Quantifying the Resilience and Vulnerability of HIV-1 Native Glycan Shield

Dense surface glycosylation on the HIV-1 envelope (Env) protein acts as a shield from the adaptive immune system. However, the molecular complexity and flexibility of glycans make experimental studies a challenge. Here we have integrated high-throughput atomistic modeling of fully glycosylated HIV-1 Env with graph theory to capture immunologically important features of the shield topology. This is the first complete all-atom model of HIV-1 Env SOSIP glycan shield that includes both oligomannose and complex glycans, providing results which are physiologically more relevant than the previous models with uniform glycosylation. This integrated approach including quantitative comparison with cryo-electron microscopy data provides hitherto unexplored details of the native shield architecture and its difference from the high-mannose glycoform. We have also derived a measure to quantify the shielding effect over the antigenic protein surface that defines regions of relative vulnerability and resilience of the shield and can be harnessed for rational immunogen design.

biophysics↗

bioRxiv2019

HIV-1 Envelope and MPER antibody structures in lipid assemblies

Structural and functional studies of HIV Env as a transmembrane protein have long been complicated by challenges associated with inherent flexibility of the molecule and the membrane-embedded hydrophobic regions. Thus, most structural studies have utilized soluble forms where the regions C-terminal to the ectodomain are deleted. Here, we present approaches for incorporating full-length, wild-type HIV-1 Env, as well as C-terminally truncated and stabilized versions, into lipid assemblies, providing a modular platform for Env structural studies by single particle electron microscopy. We reconstituted a full-length Env clone into a nanodisc with MSP1D1 scaffold, complexed it with an MPER targeting antibody 10E8, and structurally defined the full quaternary epitope of 10E8 consisting of lipid, MPER and ectodomain contacts. By aligning this and other Env-MPER antibody complex reconstructions with the lipid bilayer, we observe evidence of Env tilting as part of the neutralization mechanism for MPER-targeting antibodies. We also adapted the platform toward vaccine design purposes by introducing stabilizing mutations that allow purification of unliganded Env with peptidisc scaffold.

immunology↗

bioRxiv2019

Visualization of the HIV-1 Env Glycan Shield Across Scales

The dense array of N-linked glycans on the HIV-1 Envelope Glycoprotein (Env), known as the "glycan shield", is a key determinant of immunogenicity, yet intrinsic heterogeneity confounds typical structure-function analysis. Here we present an integrated approach of single-particle electron cryomicroscopy (cryo-EM), computational modeling, and site-specific mass-spectrometry (MS) to probe glycan shield structure and behavior at multiple levels. We found that dynamics lead to an extensive network of inter-glycan interactions that drive the formation of higher-order structure within the glycan shield. This structure defines diffuse boundaries between buried and exposed protein surface and creates a mapping of potentially immunogenic sites on Env. Analysis of Env expressed in different cell lines revealed how cryo-EM can detect subtle changes in glycan occupancy, composition, and dynamics that impact glycan shield structure and epitope accessibility. Importantly, this identified unforeseen changes in the glycan shield of Env obtained from expression in the same CHO cell line used for GMP production. Finally, by capturing the enzymatic deglycosylation of Env in a time-resolved manner we found that highly connected glycan clusters are resistant to digestion and help stabilize the pre-fusion trimer, suggesting the glycan shield may function beyond immune evasion. Significance StatementThe HIV-1 Env "glycan shield" masks the surface of the protein from immune recognition, yet intrinsic heterogeneity defies a typical structure-function description. Using a complementary approach of cryo-EM, computational modeling, and mass-spectrometry we show how heterogeneity and dynamics affect glycan shield structure across scales. Our combined approach facilitated the development of new cryo-EM data analysis methods and allowed for validation of models against experiment. Comparison of Env across a range of glycosylation states revealed how subtle differences in composition impact glycan shield structure and affect the accessibility of epitopes on the surface. Finally, time-resolved cryo-EM experiments uncovered how highly connected glycan clusters help stabilize the pre-fusion trimer, suggesting the glycan shield may function beyond immune evasion.

biophysics↗