Direct Visualization of the Conformational Dynamics of Single Influenza Hemagglutinin Trimers

Das, Dibyendu; Govindan, Ramesh; Nikić, Ivana; Krammer, Florian; Lemke, Edward A.; Munro, James B.

doi:10.1016/j.cell.2018.05.050

Cited by 125 publications

(192 citation statements)

References 56 publications

(93 reference statements)

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“…The inferred movements of the HA heads to enable antibody H7.5 binding suggest that the HA1 head is "breathing", and this motion is reminiscent of conformational masking described for the HIV envelope glycoprotein (23)(24)(25). Indeed, a very recent study using single molecule FRET observed that H5 HA undergoes reversible conformational changes (26).…”

Section: Discussionmentioning

confidence: 93%

Potent anti-influenza H7 human monoclonal antibody induces separation of hemagglutinin receptor binding head domains

Turner

Pallesen

Lang

et al. 2018

Preprint

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Seasonal influenza virus infections can cause significant morbidity and mortality, but the threat from emergence of a new pandemic influenza strain might have potentially even more devastating consequences. As such, there is intense interest in isolating and characterizing potent neutralizing antibodies that target the hemagglutinin (HA) viral surface glycoprotein. Here, we use cryo-electron microscopy to decipher the mechanism of action of a potent HA head-directed monoclonal antibody bound to an influenza H7 HA. The epitope of the antibody is not solvent accessible in the compact, pre-fusion conformation that typifies all HA structures to date.Instead, the antibody binds between HA head protomers to an epitope that must be partly or transiently exposed in the pre-fusion conformation. The "breathing" of the HA protomers is implied by the exposure of this epitope, which is consistent with metastability of class I fusion proteins. This structure likely therefore represents an early structural intermediate in the viral fusion process. Understanding the extent of transient exposure of conserved neutralizing epitopes also may lead to new opportunities to combat influenza that have not been appreciated previously. Author SummaryA transiently exposed epitope on influenza H7 hemagglutinin represents a new target for neutralizing antibodies.

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Section: Discussionmentioning

confidence: 93%

Potent anti-influenza H7 human monoclonal antibody induces separation of hemagglutinin receptor binding head domains

Turner

Pallesen

Lang

et al. 2018

Preprint

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“…Once triggered, this energetic imbalance ultimately results in the fusion protein undergoing an irreversible transition to the post-fusion state. Indeed many early pioneering studies on influenza HA led to the development of the "spring-loaded" mechanistic model for viral membrane fusion that is the prevailing way in which these machines are considered to function [21][22][23][24][25][26][27][28][29][30][31][32]. In this model, type I fusion proteins function analogous to taut springs that are poised in a high energy state that, once triggered, rapidly and irreversibly "spring" or refold to the low-energy, post-fusion state.…”

mentioning

confidence: 99%

New Biophysical Approaches Reveal the Dynamics and Mechanics of Type I Viral Fusion Machinery and Their Interplay with Membranes

Benhaim

Lee

2020

Viruses

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Protein-mediated membrane fusion is a highly regulated biological process essential for cellular and organismal functions and infection by enveloped viruses. During viral entry the membrane fusion reaction is catalyzed by specialized protein machinery on the viral surface. These viral fusion proteins undergo a series of dramatic structural changes during membrane fusion where they engage, remodel, and ultimately fuse with the host membrane. The structural and dynamic nature of these conformational changes and their impact on the membranes have long-eluded characterization. Recent advances in structural and biophysical methodologies have enabled researchers to directly observe viral fusion proteins as they carry out their functions during membrane fusion. Here we review the structure and function of type I viral fusion proteins and mechanisms of protein-mediated membrane fusion. We highlight how recent technological advances and new biophysical approaches are providing unprecedented new insight into the membrane fusion reaction.Viruses 2020, 12, 413 2 of 21 built. Type II fusion proteins are found in viruses including flaviviruses and alphaviruses such as Dengue, Zika, and Chikungunya viruses, and even have been identified in eukaryotic cell-cell fusion systems [9][10][11][12]. Type III fusion proteins are found in rhabdoviruses (such as Rabies and Vesicular Stomatatis virus G glycoproteins), herpesviruses (Herpes Simplex virus 1 gB protein), as well as baculovirus [12][13][14][15][16]. While the individual folds exhibited by these classes are completely different, they share common functional traits in that all adopt a pre-fusion conformation prior to activation in which one terminus of the protein is anchored in the virus membrane by a transmembrane domain and a second membrane active component, either a fusion peptide or loop, is sequestered from interacting with membranes ( Figure 1) [12]. A trigger or set of triggers, such as exposure to low pH in endosomes or receptor binding, spurs the machinery to reorganize into a post-fusion state in which the two membrane active components are colocalized.Until recently, static structures of the pre-and post-fusion states of isolated fusion protein ectodomains and biochemical or spectroscopic measurements were the primary pieces of information that informed our models of membrane fusion. While the static structures provide defined endpoints for the conformational change that drives membrane fusion, they do not tell us how these conformational changes occur or how these proteins interact with and perturb the lipid membrane during fusion. Likewise, fluorescence spectroscopy and circular dichroism studies have shown that HA-fusion activation leads to population of discernable intermediates rather than transitioning directly and irreversibly from pre-to post-fusion states [17][18][19][20]. These studies show that not all HAs respond to activation in the same way and some require different pH conditions to fully activate [20]. While such studies provided valuable information...

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“…As shown above, CSOP has the potential to be a broadly useful tool for investigating diverse biological processes including protein isoform changes during cell differentiation (51), modifications in glycan height during cancer progression (26), and kinetics of protein conformation changes (52). Ongoing developments in protein labeling strategies using antibodies and site-specific chemical labels have the potential to improve the ability of CSOP to quantify the height and flexibility of cell surface molecules and their physiological impacts.…”

Section: Discussionmentioning

confidence: 99%

Molecular height measurement by cell surface optical profilometry (CSOP)

Son

Takatori

Belardi

et al. 2020

Preprint

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The physical dimensions of proteins and glycans on cell surfaces can critically affect cell function, for example by preventing close contact between cells and limiting receptor accessibility. However, high-resolution measurements of molecular heights on native cell membranes have been difficult to obtain. Here we present a simple and rapid method that achieves nanometer height resolution by localizing fluorophores at the tip and base of cell surface molecules and determining their separation by radially averaging across many molecules. We use this method, which we call cell surface optical profilometry (CSOP), to quantify height of key multi-domain proteins on a model macrophage and cancer cell, as well as to capture average protein and glycan heights on native cell membranes. We show that average height of a protein is significantly smaller than its contour length due to thermally driven bending and rotation on the membrane and that height strongly depends on local surface and solution conditions. We find that average height increases with cell surface molecular crowding, while it decreases with solution crowding by solutes, both of which we confirm with molecular dynamics simulations. We also use experiments and simulations to determine the height of an epitope based on the location of an antibody, which allows CSOP to profile various proteins and glycans on a native cell surface using antibodies and lectins. This versatile method for profiling cell surfaces has the potential to advance understanding of the molecular landscape of cells and its role in cell function.

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Direct Visualization of the Conformational Dynamics of Single Influenza Hemagglutinin Trimers

Cited by 125 publications

References 56 publications

Potent anti-influenza H7 human monoclonal antibody induces separation of hemagglutinin receptor binding head domains

Potent anti-influenza H7 human monoclonal antibody induces separation of hemagglutinin receptor binding head domains

New Biophysical Approaches Reveal the Dynamics and Mechanics of Type I Viral Fusion Machinery and Their Interplay with Membranes

Molecular height measurement by cell surface optical profilometry (CSOP)

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