Allosteric mechanism of membrane fusion activation in a herpesvirus.

Herpesviridae infect nearly all humans for life, causing diseases that range from painful to life-threatening 1 . These viruses penetrate cells by employing a complex apparatus composed of separate receptor-binding, signal-transmitting, and membrane-fusing components 2 . But how these components coordinate their functions is unknown. Here, we determined the 4.19-angstrom cryoEM reconstruction of the central signal-transmitting component from herpes simplex virus 2, the gH/gL complex, in its elusive pre-activation state. Analysis of the continuum of conformational ensembles observed in cryoEM data revealed a series of structural rearrangements in gH/gL that allosterically transmit the fusion-triggering signal from the receptor-binding glycoprotein gD to the membrane fusogen gB. Furthermore, we identified a structural "switch" element in gH/gL that refolds and flips 180 degrees during the transition from pre-activation to activated form. Conservation of this "switch" in gH/gL homologs suggests that the proposed fusion triggering mechanism may apply to all Herpesviridae and points to a new target for subunit-based vaccines and treatment efforts.

Structural basis of ligand specificity and channel activation in an insect gustatory receptor.

Gustatory receptors (GRs) are critical for insect chemosensation and are potential targets for controlling pests and disease vectors, making their structural investigation a vital step toward such applications. We present structures of Bombyx mori Gr9 (BmGr9), a fructose-gated cation channel, in agonist-free and fructose-bound states. BmGr9 forms a tetramer similar to distantly related insect odorant receptors (ORs). Upon fructose binding, BmGr9's channel gate opens through helix S7b movements. In contrast to ORs, BmGr9's ligand-binding pocket, shaped by a kinked helix S4 and a shorter extracellular S3-S4 loop, is larger and solvent accessible in both agonist-free and fructose-bound states. Also, unlike ORs, fructose binding by BmGr9 involves helix S5 and a pocket lined with aromatic and polar residues. Structure-based sequence alignments reveal distinct patterns of ligand-binding pocket residue conservation in GR subfamilies associated with different ligand classes. These data provide insight into the molecular basis of GR ligand specificity and function.

Mechanism of autocatalytic activation during proteasome assembly.

Many large molecular machines are too elaborate to assemble spontaneously and are built through ordered pathways orchestrated by dedicated chaperones. During assembly of the core particle (CP) of the proteasome, where protein degradation occurs, its six active sites are simultaneously activated via cleavage of N-terminal propeptides. Such activation is autocatalytic and coupled to fusion of two half-CP intermediates, which protects cells by preventing activation until enclosure of the active sites within the CP interior. Here we uncover key mechanistic aspects of autocatalytic activation, which proceeds through alignment of the ß5 and ß2 catalytic triad residues, respectively, with these triads being misaligned before fusion. This mechanism contrasts with most other zymogens, in which catalytic centers are preformed. Our data also clarify the mechanism by which individual subunits can be added in a precise, temporally ordered manner. This work informs two decades-old mysteries in the proteasome field, with broader implications for protease biology and multisubunit complex assembly.

Visualizing chaperone-mediated multistep assembly of the human 20S proteasome.

Dedicated assembly factors orchestrate the stepwise production of many molecular machines, including the 28-subunit proteasome core particle (CP) that mediates protein degradation. Here we report cryo-electron microscopy reconstructions of seven recombinant human subcomplexes that visualize all five chaperones and the three active site propeptides across a wide swath of the assembly pathway. Comparison of these chaperone-bound intermediates and a matching mature CP reveals molecular mechanisms determining the order of successive subunit additions, as well as how proteasome subcomplexes and assembly factors structurally adapt upon progressive subunit incorporation to stabilize intermediates, facilitate the formation of subsequent intermediates and ultimately rearrange to coordinate proteolytic activation with gated access to active sites. This work establishes a methodologic approach for structural analysis of multiprotein complex assembly intermediates, illuminates specific functions of assembly factors and reveals conceptual principles underlying human proteasome biogenesis, thus providing an explanation for many previous biochemical and genetic observations.

Visualizing chaperone-mediated multistep assembly of the human 20S proteasome.

Dedicated assembly factors orchestrate stepwise production of many molecular machines, including the 28-subunit proteasome core particle (CP) that mediates protein degradation. Here, we report cryo-EM reconstructions of seven recombinant human subcomplexes that visualize all five chaperones and the three active site propeptides across a wide swath of the assembly pathway. Comparison of these chaperone-bound intermediates and a matching mature CP reveals molecular mechanisms determining the order of successive subunit additions, and how proteasome subcomplexes and assembly factors structurally adapt upon progressive subunit incorporation to stabilize intermediates, facilitate the formation of subsequent intermediates, and ultimately rearrange to coordinate proteolytic activation with gated access to active sites. The structural findings reported here explain many previous biochemical and genetic observations. This work establishes a methodologic approach for structural analysis of multiprotein complex assembly intermediates, illuminates specific functions of assembly factors, and reveals conceptual principles underlying human proteasome biogenesis.

Protective human antibodies against a conserved epitope in pre- and postfusion influenza hemagglutinin.

Phylogenetically and antigenically distinct influenza A and B viruses (IAV and IBV) circulate in human populations, causing widespread morbidity. Antibodies (Abs) that bind epitopes conserved in both IAV and IBV hemagglutinins (HAs) could protect against disease by diverse virus subtypes. Only one reported HA Ab, isolated from a combinatorial display library, protects against both IAV and IBV. Thus, there has been so far no information on the likelihood of finding naturally occurring human Abs that bind HAs of diverse IAV subtypes and IBV lineages. We have now recovered from several unrelated human donors five clonal Abs that bind a conserved epitope preferentially exposed in the postfusion conformation of IAV and IVB HA2. These Abs lack neutralizing activity in vitro but in mice provide strong, IgG subtype-dependent protection against lethal IAV and IBV infections. Strategies to elicit similar Abs routinely might contribute to more effective influenza vaccines.

Structure of the preholoproteasome reveals late steps in proteasome core particle biogenesis.

Assembly of the proteasome's core particle (CP), a barrel-shaped chamber of four stacked rings, requires five chaperones and five subunit propeptides. Fusion of two half-CP precursors yields a complete structure but remains immature until active site maturation. Here, using Saccharomyces cerevisiae, we report a high-resolution cryogenic electron microscopy structure of preholoproteasome, a post-fusion assembly intermediate. Our data reveal how CP midline-spanning interactions induce local changes in structure, facilitating maturation. Unexpectedly, we find that cleavage may not be sufficient for propeptide release, as residual interactions with chaperones such as Ump1 hold them in place. We evaluated previous models proposing that dynamic conformational changes in chaperones drive CP fusion and autocatalytic activation by comparing preholoproteasome to pre-fusion intermediates. Instead, the data suggest a scaffolding role for the chaperones Ump1 and Pba1/Pba2. Our data clarify key aspects of CP assembly, suggest that undiscovered mechanisms exist to explain CP fusion/activation, and have relevance for diseases of defective CP biogenesis.

Structural basis of colibactin activation by the ClbP peptidase.

Colibactin, a DNA cross-linking agent produced by gut bacteria, is implicated in colorectal cancer. Its biosynthesis uses a prodrug resistance mechanism: a non-toxic precursor assembled in the cytoplasm is activated after export to the periplasm. This activation is mediated by ClbP, an inner-membrane peptidase with an N-terminal periplasmic catalytic domain and a C-terminal three-helix transmembrane domain. Although the transmembrane domain is required for colibactin activation, its role in catalysis is unclear. Our structure of full-length ClbP bound to a product analog reveals an interdomain interface important for substrate binding and enzyme stability and interactions that explain the selectivity of ClbP for the N-acyl-D-asparagine prodrug motif. Based on structural and biochemical evidence, we propose that ClbP dimerizes to form an extended substrate-binding site that can accommodate a pseudodimeric precolibactin with its two terminal prodrug motifs in the two ClbP active sites, thus enabling the coordinated activation of both electrophilic warheads.

Structure of an insect gustatory receptor.

Gustatory Receptors (GRs) are critical for insect chemosensation and are potential targets for controlling pests and disease vectors. However, GR structures have not been experimentally determined. We present structures of Bombyx mori Gr9 (BmGr9), a fructose-gated cation channel, in agonist-free and fructose-bound states. BmGr9 forms a tetramer similar to distantly related insect Olfactory Receptors (ORs). Upon fructose binding, BmGr9's ion channel gate opens through helix S7b movements. In contrast to ORs, BmGR9's ligand-binding pocket, shaped by a kinked helix S4 and a shorter extracellular S3-S4 loop, is larger and solvent accessible in both agonist-free and fructose-bound states. Also unlike ORs, fructose binding by BmGr9 involves helix S5 and a binding pocket lined with aromatic and polar residues. Structure-based sequence alignments reveal distinct patterns of ligand-binding pocket residue conservation in GR subfamilies associated with distinct ligand classes. These data provide insight into the molecular basis of GR ligand specificity and function.

Recent advances in infectious disease research using cryo-electron tomography.

With the increasing spread of infectious diseases worldwide, there is an urgent need for novel strategies to combat them. Cryogenic sample electron microscopy (cryo-EM) techniques, particularly electron tomography (cryo-ET), have revolutionized the field of infectious disease research by enabling multiscale observation of biological structures in a near-native state. This review highlights the recent advances in infectious disease research using cryo-ET and discusses the potential of this structural biology technique to help discover mechanisms of infection in native environments and guiding in the right direction for future drug discovery.

Practices for running a research-oriented shared cryo-EM facility.

The Harvard Cryo-Electron Microscopy Center for Structural Biology, which was formed as a consortium between Harvard Medical School, Boston Children's Hospital, Dana-Farber Cancer Institute, and Massachusetts General Hospital, serves both academic and commercial users in the greater Harvard community. The facility strives to optimize research productivity while training users to become expert electron microscopists. These two tasks may be at odds and require careful balance to keep research projects moving forward while still allowing trainees to develop independence and expertise. This article presents the model developed at Harvard Medical School for running a research-oriented cryo-EM facility. Being a research-oriented facility begins with training in cryo-sample preparation on a trainee's own sample, ideally producing grids that can be screened and optimized on the Talos Arctica via multiple established pipelines. The first option, staff assisted screening, requires no user experience and a staff member provides instant feedback about the suitability of the sample for cryo-EM investigation and discusses potential strategies for sample optimization. Another option, rapid access, allows users short sessions to screen samples and introductory training for basic microscope operation. Once a sample reaches the stage where data collection is warranted, new users are trained on setting up data collection for themselves on either the Talos Arctica or Titan Krios microscope until independence is established. By providing incremental training and screening pipelines, the bottleneck of sample preparation can be overcome in parallel with developing skills as an electron microscopist. This approach allows for the development of expertise without hindering breakthroughs in key research areas.

Yeast PI31 inhibits the proteasome by a direct multisite mechanism.

Proteasome inhibitors are widely used as therapeutics and research tools, and typically target one of the three active sites, each present twice in the proteasome complex. An endogeneous proteasome inhibitor, PI31, was identified 30 years ago, but its inhibitory mechanism has remained unclear. Here, we identify the mechanism of Saccharomyces cerevisiae PI31, also known as Fub1. Using cryo-electron microscopy (cryo-EM), we show that the conserved carboxy-terminal domain of Fub1 is present inside the proteasome's barrel-shaped core particle (CP), where it simultaneously interacts with all six active sites. Targeted mutations of Fub1 disrupt proteasome inhibition at one active site, while leaving the other sites unaffected. Fub1 itself evades degradation through distinct mechanisms at each active site. The gate that allows substrates to access the CP is constitutively closed, and Fub1 is enriched in mutant CPs with an abnormally open gate, suggesting that Fub1 may function to neutralize aberrant proteasomes, thereby ensuring the fidelity of proteasome-mediated protein degradation.

Chaperone-mediated assembly of the proteasome core particle - recent developments and structural insights.

Much of cellular activity is mediated by large multisubunit complexes. However, many of these complexes are too complicated to assemble spontaneously. Instead, their biogenesis is facilitated by dedicated chaperone proteins, which are themselves excluded from the final product. This is the case for the proteasome, a ubiquitous and highly conserved cellular regulator that mediates most selective intracellular protein degradation in eukaryotes. The proteasome consists of two subcomplexes: the core particle (CP), where proteolysis occurs, and the regulatory particle (RP), which controls substrate access to the CP. Ten chaperones function in proteasome biogenesis. Here, we review the pathway of CP biogenesis, which requires five of these chaperones and proceeds through a highly ordered multistep pathway. We focus on recent advances in our understanding of CP assembly, with an emphasis on structural insights. This pathway of CP biogenesis represents one of the most dramatic examples of chaperone-mediated assembly and provides a paradigm for understanding how large multisubunit complexes can be produced.

Mechanism of proteasome gate modulation by assembly chaperones Pba1 and Pba2.

The active sites of the proteasome are housed within its central core particle (CP), a barrel-shaped chamber of four stacked heptameric rings, and access of substrates to the CP interior is mediated by gates at either axial end. These gates are constitutively closed and may be opened by the regulatory particle (RP), which binds the CP and facilitates substrate degradation. We recently showed that the heterodimeric CP assembly chaperones Pba1/2 also mediate gate opening through an unexpected structural arrangement that facilitates the insertion of the N terminus of Pba1 into the CP interior; however, the full mechanism of Pba1/2-mediated gate opening is unclear. Here, we report a detailed analysis of CP gate modulation by Pba1/2. The clustering of key residues at the interface between neighboring α-subunits is a critical feature of RP-mediated gate opening, and we find that Pba1/2 recapitulate this strategy. Unlike RP, which inserts at six α-subunit interfaces, Pba1/2 insert at only two α-subunit interfaces. Nevertheless, Pba1/2 are able to regulate six of the seven interfacial clusters, largely through direct interactions. The N terminus of Pba1 also physically interacts with the center of the gate, disrupting the intersubunit contacts that maintain the closed state. This novel mechanism of gate modulation appears to be unique to Pba1/2 and therefore likely occurs only during proteasome assembly. Our data suggest that release of Pba1/2 at the conclusion of assembly is what allows the nascent CP to assume its mature gate conformation, which is primarily closed, until activated by RP.

Membrane fusion and immune evasion by the spike protein of SARS-CoV-2 Delta variant.

The Delta variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has outcompeted previously prevalent variants and become a dominant strain worldwide. We report the structure, function, and antigenicity of its full-length spike (S) trimer as well as those of the Gamma and Kappa variants, and compare their characteristics with the G614, Alpha, and Beta variants. Delta S can fuse membranes more efficiently at low levels of cellular receptor angiotensin converting enzyme 2 (ACE2), and its pseudotyped viruses infect target cells substantially faster than the other five variants, possibly accounting for its heightened transmissibility. Each variant shows different rearrangement of the antigenic surface of the amino-terminal domain of the S protein but only makes produces changes in the receptor binding domain (RBD), making the RBD a better target for therapeutic antibodies.

Membrane fusion and immune evasion by the spike protein of SARS-CoV-2 Delta variant.

The Delta variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has outcompeted previously prevalent variants and become a dominant strain worldwide. We report here structure, function and antigenicity of its full-length spike (S) trimer in comparison with those of other variants, including Gamma, Kappa, and previously characterized Alpha and Beta. Delta S can fuse membranes more efficiently at low levels of cellular receptor ACE2 and its pseudotyped viruses infect target cells substantially faster than all other variants tested, possibly accounting for its heightened transmissibility. Mutations of each variant rearrange the antigenic surface of the N-terminal domain of the S protein in a unique way, but only cause local changes in the receptor-binding domain, consistent with greater resistance particular to neutralizing antibodies. These results advance our molecular understanding of distinct properties of these viruses and may guide intervention strategies.

Structural basis for enhanced infectivity and immune evasion of SARS-CoV-2 variants.

Several fast-spreading variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have become the dominant circulating strains in the COVID-19 pandemic. We report here cryo-electron microscopy structures of the full-length spike (S) trimers of the B.1.1.7 and B.1.351 variants, as well as their biochemical and antigenic properties. Amino acid substitutions in the B.1.1.7 protein increase both the accessibility of its receptor binding domain and the binding affinity for receptor angiotensin-converting enzyme 2 (ACE2). The enhanced receptor engagement may account for the increased transmissibility. The B.1.351 variant has evolved to reshape antigenic surfaces of the major neutralizing sites on the S protein, making it resistant to some potent neutralizing antibodies. These findings provide structural details on how SARS-CoV-2 has evolved to enhance viral fitness and immune evasion.

Structures of chaperone-associated assembly intermediates reveal coordinated mechanisms of proteasome biogenesis.

The proteasome mediates most selective protein degradation. Proteolysis occurs within the 20S core particle (CP), a barrel-shaped chamber with an α7ß7ß7α7 configuration. CP biogenesis proceeds through an ordered multistep pathway requiring five chaperones, Pba1-4 and Ump1. Using Saccharomyces cerevisiae, we report high-resolution structures of CP assembly intermediates by cryogenic-electron microscopy. The first structure corresponds to the 13S particle, which consists of a complete α-ring, partial ß-ring (ß2-4), Ump1 and Pba1/2. The second structure contains two additional subunits (ß5-6) and represents a later pre-15S intermediate. These structures reveal the architecture and positions of Ump1 and ß2/ß5 propeptides, with important implications for their functions. Unexpectedly, Pba1's N terminus extends through an open CP pore, accessing the CP interior to contact Ump1 and the ß5 propeptide. These results reveal how the coordinated activity of Ump1, Pba1 and the active site propeptides orchestrate key aspects of CP assembly.

Structural basis for enhanced infectivity and immune evasion of SARS-CoV-2 variants.

Several fast-spreading variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have become the dominant circulating strains that continue to fuel the COVID-19 pandemic despite intensive vaccination efforts throughout the world. We report here cryo-EM structures of the full-length spike (S) trimers of the B.1.1.7 and B.1.351 variants, as well as their biochemical and antigenic properties. Mutations in the B.1.1.7 protein increase the accessibility of its receptor binding domain and also the binding affinity for receptor angiotensin-converting enzyme 2 (ACE2). The enhanced receptor engagement can account for the increased transmissibility and risk of mortality as the variant may begin to infect efficiently infect additional cell types expressing low levels of ACE2. The B.1.351 variant has evolved to reshape antigenic surfaces of the major neutralizing sites on the S protein, rendering complete resistance to some potent neutralizing antibodies. These findings provide structural details on how the wide spread of SARS-CoV-2 enables rapid evolution to enhance viral fitness and immune evasion. They may guide intervention strategies to control the pandemic.

Structural impact on SARS-CoV-2 spike protein by D614G substitution.

Substitution for aspartic acid (D) by glycine (G) at position 614 in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) appears to facilitate rapid viral spread. The G614 strain and its recent variants are now the dominant circulating forms. Here, we report cryo-electron microscopy structures of a full-length G614 S trimer, which adopts three distinct prefusion conformations that differ primarily by the position of one receptor-binding domain. A loop disordered in the D614 S trimer wedges between domains within a protomer in the G614 spike. This added interaction appears to prevent premature dissociation of the G614 trimer-effectively increasing the number of functional spikes and enhancing infectivity-and to modulate structural rearrangements for membrane fusion. These findings extend our understanding of viral entry and suggest an improved immunogen for vaccine development.