Cryo-ET of Env on intact HIV virions reveals structural variation and positioning on the Gag lattice.

HIV-1 Env mediates viral entry into host cells and is the sole target for neutralizing antibodies. However, Env structure and organization in its native virion context has eluded detailed characterization. Here, we used cryo-electron tomography to analyze Env in mature and immature HIV-1 particles. Immature particles showed distinct Env positioning relative to the underlying Gag lattice, providing insights into long-standing questions about Env incorporation. A 9.1-Å sub-tomogram-averaged reconstruction of virion-bound Env in conjunction with structural mass spectrometry revealed unexpected features, including a variable central core of the gp41 subunit, heterogeneous glycosylation between protomers, and a flexible stalk that allows Env tilting and variable exposure of neutralizing epitopes. Together, our results provide an integrative understanding of HIV assembly and structural variation in Env antigen presentation.

Induction of Potent Neutralizing Antibody Responses by a Designed Protein Nanoparticle Vaccine for Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is a worldwide public health concern for which no vaccine is available. Elucidation of the prefusion structure of the RSV F glycoprotein and its identification as the main target of neutralizing antibodies have provided new opportunities for development of an effective vaccine. Here, we describe the structure-based design of a self-assembling protein nanoparticle presenting a prefusion-stabilized variant of the F glycoprotein trimer (DS-Cav1) in a repetitive array on the nanoparticle exterior. The two-component nature of the nanoparticle scaffold enabled the production of highly ordered, monodisperse immunogens that display DS-Cav1 at controllable density. In mice and nonhuman primates, the full-valency nanoparticle immunogen displaying 20 DS-Cav1 trimers induced neutralizing antibody responses ∼10-fold higher than trimeric DS-Cav1. These results motivate continued development of this promising nanoparticle RSV vaccine candidate and establish computationally designed two-component nanoparticles as a robust and customizable platform for structure-based vaccine design.

Single-Molecule Analysis of a Viral Fusion Protein Illuminates a Fusion-Active Intermediate State.

The influenza virus hemagglutinin (HA) fusion glycoprotein mediates viral entry into host cells through its receptor binding and membrane fusion activities. In this issue of Cell, Das et al. use single-molecule Förster resonance energy transfer (smFRET) to monitor HA conformational dynamics. Their study reveals this prototypical class I fusion protein to be a highly dynamic molecule capable of reversibly sampling multiple states, including on-pathway fusion intermediates between pre-fusion and post-fusion endpoints. These findings challenge long-held ideas for how HA functions and move the field closer to obtaining a mechanistic understanding of how class I fusion proteins mediate membrane fusion.

Structural dynamics reveal subtype-specific activation and inhibition of influenza virus hemagglutinin.

Influenza hemagglutinin (HA) is a prototypical class 1 viral entry glycoprotein, responsible for mediating receptor binding and membrane fusion. Structures of its prefusion and postfusion forms, embodying the beginning and endpoints of the fusion pathway, have been extensively characterized. Studies probing HA dynamics during fusion have begun to identify intermediate states along the pathway, enhancing our understanding of how HA becomes activated and traverses its conformational pathway to complete fusion. HA is also the most variable, rapidly evolving part of influenza virus, and it is not known whether mechanisms of its activation and fusion are conserved across divergent viral subtypes. Here, we apply hydrogen-deuterium exchange mass spectrometry to compare fusion activation in two subtypes of HA, H1 and H3. Our data reveal subtype-specific behavior in the regions of HA that undergo structural rearrangement during fusion, including the fusion peptide and HA1/HA2 interface. In the presence of an antibody that inhibits the conformational change (FI6v3), we observe that acid-induced dynamic changes near the epitope are dampened, but the degree of protection at the fusion peptide is different for the two subtypes investigated. These results thus provide new insights into variation in the mechanisms of influenza HA's dynamic activation and its inhibition.

Structural dynamics reveal isolate-specific differences at neutralization epitopes on HIV Env.

The envelope glycoprotein (Env) is the sole target for neutralizing antibodies against HIV and the most rapidly evolving, variable part of the virus. High-resolution structures of Env trimers captured in the pre-fusion, closed conformation have revealed a high degree of structural similarity across diverse isolates. Biophysical data, however, indicate that Env is highly dynamic, and the level of dynamics and conformational sampling is believed to vary dramatically between HIV isolates. Dynamic differences likely influence neutralization sensitivity, receptor activation, and overall trimer stability. Here, using hydrogen/deuterium-exchange mass spectrometry (HDX-MS), we have mapped local dynamics across native-like Env SOSIP trimers from diverse isolates. We show that significant differences in epitope order are observed across most sites targeted by broadly neutralizing antibodies. We also observe isolate-dependent conformational switching that occurs over a broad range of timescales. Lastly, we report that hyper-stabilizing mutations that dampen dynamics in some isolates have little effect on others.

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

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.

Bridging protein structure, dynamics, and function using hydrogen/deuterium-exchange mass spectrometry.

Much of our understanding of protein structure and mechanistic function has been derived from static high-resolution structures. As structural biology has continued to evolve it has become clear that high-resolution structures alone are unable to fully capture the mechanistic basis for protein structure and function in solution. Recently Hydrogen/Deuterium-exchange Mass Spectrometry (HDX-MS) has developed into a powerful and versatile tool for structural biologists that provides novel insights into protein structure and function. HDX-MS enables direct monitoring of a protein's structural fluctuations and conformational changes under native conditions in solution even as it is carrying out its functions. In this review, we focus on the use of HDX-MS to monitor these dynamic changes in proteins. We examine how HDX-MS has been applied to study protein structure and function in systems ranging from large, complex assemblies to intrinsically disordered proteins, and we discuss its use in probing conformational changes during protein folding and catalytic function. STATEMENT FOR A BROAD AUDIENCE: The biophysical and structural characterization of proteins provides novel insight into their functionalities. Protein motions, ranging from small scale local fluctuations to larger concerted structural rearrangements, often determine protein function. Hydrogen/Deuterium-exchange Mass Spectrometry (HDX-MS) has proven a powerful biophysical tool capable of probing changes in protein structure and dynamic protein motions that are often invisible to most other techniques.

Tracking Higher Order Protein Structure by Hydrogen-Deuterium Exchange Mass Spectrometry.

BACKGROUND: Structural biology has provided a fundamental understanding of protein structure and mechanistic insight into their function. However, high-resolution structures alone are insufficient for a complete understanding of protein behavior. Higher energy conformations, conformational changes, and subtle structural fluctuations that underlie the proper function of proteins are often difficult to probe using traditional structural approaches. Hydrogen/Deuterium Exchange with Mass Spectrometry (HDX-MS) provides a way to probe the accessibility of backbone amide protons under native conditions, which reports on local structural dynamics of solution protein structure that can be used to track complex structural rearrangements that occur in the course of a protein's function. CONCLUSION: In the last 20 years the advances in labeling techniques, sample preparation, instrumentation, and data analysis have enabled HDX to gain insights into very complex biological systems. Analysis of challenging targets such as membrane protein complexes is now feasible and the field is paving the way to the analysis of more and more complex systems.

De novo design of tunable, pH-driven conformational changes.

The ability of naturally occurring proteins to change conformation in response to environmental changes is critical to biological function. Although there have been advances in the de novo design of stable proteins with a single, deep free-energy minimum, the design of conformational switches remains challenging. We present a general strategy to design pH-responsive protein conformational changes by precisely preorganizing histidine residues in buried hydrogen-bond networks. We design homotrimers and heterodimers that are stable above pH 6.5 but undergo cooperative, large-scale conformational changes when the pH is lowered and electrostatic and steric repulsion builds up as the network histidine residues become protonated. The transition pH and cooperativity can be controlled through the number of histidine-containing networks and the strength of the surrounding hydrophobic interactions. Upon disassembly, the designed proteins disrupt lipid membranes both in vitro and after being endocytosed in mammalian cells. Our results demonstrate that environmentally triggered conformational changes can now be programmed by de novo protein design.