Mimotope discovery as a tool to design a vaccine against Zika and dengue viruses.

Vaccine development against dengue virus is challenging because of the antibody-dependent enhancement of infection (ADE), which causes severe disease. Consecutive infections by Zika (ZIKV) and/or dengue viruses (DENV), or vaccination can predispose to ADE. Current vaccines and vaccine candidates contain the complete envelope viral protein, with epitopes that can raise antibodies causing ADE. We used the envelope dimer epitope (EDE), which induces neutralizing antibodies that do not elicit ADE, to design a vaccine against both flaviviruses. However, EDE is a discontinuous quaternary epitope that cannot be isolated from the E protein without other epitopes. Utilizing phage display, we selected three peptides that mimic the EDE. Free mimotopes were disordered and did not elicit an immune response. After their display on adeno-associated virus (AAV) capsids (VLP), they recovered their structure and were recognized by an EDE-specific antibody. Characterization by cryo-EM and enzyme-linked immunosorbent assay confirmed the correct display of a mimotope on the surface of the AAV VLP and its recognition by the specific antibody. Immunization with the AAV VLP displaying one of the mimotopes induced antibodies that recognized ZIKV and DENV. This work provides the basis for developing a Zika and dengue virus vaccine candidate that will not induce ADE.

Assembly of Building Blocks by Double-End-Anchored Polymers in the Dilute Regime Mediated by Hydrophobic Interactions at Controlled Distances.

Hierarchical assembly of building blocks via competing, orthogonal interactions is a hallmark of many of nature's composite materials that do not require highly specific ligand-receptor interactions. To mimic this assembly mechanism requires the development of building blocks capable of tunable interactions. In the present work, we explored the interplay between repulsive (steric and electrostatic) and attractive hydrophobic forces. The designed building blocks allow hydrophobic forces to effectively act at controlled, large distances, to create and tune the assembly of membrane-based building blocks under dilute conditions, and to affect their interactions with cellular membranes via physical cross-bridges. Specifically, we employed double-end-anchored poly(ethylene glycol)s (DEA-PEGs)-hydrophilic PEG tethers with hydrophobic tails on both ends. Using differential-interference-contrast optical microscopy, synchrotron small-angle X-ray scattering (SAXS), and cryogenic electron microscopy, we investigated the ability of DEA-PEGs to mediate assembly in the dilute regime on multiple length scales and on practical time scales. The PEG length, anchor hydrophobicity, and molar fraction of DEA-PEG molecules within a membrane strongly affect the assembly properties. Additional tuning of the intermembrane interactions can be achieved by adding repulsive interactions via PEG-lipids (steric) or cationic lipids to the DEA-PEG-mediated attractions. While the optical and electron microscopy imaging methods provided qualitative evidence of the ability of DEA-PEGs to assemble liposomes, the SAXS measurements and quantitative line-shape analysis in dilute preparations demonstrated that the ensemble average of loosely organized liposomal assemblies maintains DEA-PEG concentration-dependent tethering on defined nanometer length scales. For cationic liposome-DNA nanoparticles (CL-DNA NPs), aggregation induced by DEA-PEGs decreased internalization of NPs by cells, but tuning the DEA-PEG-induced attractions by adding repulsive steric interactions via PEG-lipids limited aggregation and increased NP uptake. Furthermore, confocal microscopy imaging together with colocalization studies with Rab11 and LysoTracker as markers of intracellular pathways showed that modifying CL-DNA NPs with DEA-PEGs alters their interactions with the plasma and endosomal membranes.

Cryo-EM Structures and Regulation of Arabinofuranosyltransferase AftD from Mycobacteria.

Mycobacterium tuberculosis causes tuberculosis, a disease that kills over 1 million people each year. Its cell envelope is a common antibiotic target and has a unique structure due, in part, to two lipidated polysaccharides-arabinogalactan and lipoarabinomannan. Arabinofuranosyltransferase D (AftD) is an essential enzyme involved in assembling these glycolipids. We present the 2.9-Å resolution structure of M. abscessus AftD, determined by single-particle cryo-electron microscopy. AftD has a conserved GT-C glycosyltransferase fold and three carbohydrate-binding modules. Glycan array analysis shows that AftD binds complex arabinose glycans. Additionally, AftD is non-covalently complexed with an acyl carrier protein (ACP). 3.4- and 3.5-Å structures of a mutant with impaired ACP binding reveal a conformational change, suggesting that ACP may regulate AftD function. Mutagenesis experiments using a conditional knockout constructed in M. smegmatis confirm the essentiality of the putative active site and the ACP binding for AftD function.

PEGylation of Paclitaxel-Loaded Cationic Liposomes Drives Steric Stabilization of Bicelles and Vesicles thereby Enhancing Delivery and Cytotoxicity to Human Cancer Cells.

Poly(ethylene glycol) (PEG) is a polymer used widely in drug delivery to create "stealth" nanoparticles (NPs); PEG coatings suppress NP detection and clearance by the immune system and beneficially increase NP circulation time in vivo. However, NP PEGylation typically obstructs cell attachment and uptake in vitro compared to the uncoated equivalent. Here, we report on a cationic liposome (CL) NP system loaded with the hydrophobic cancer drug paclitaxel (PTX) in which PEGylation (i.e., PEG-CLPTX NPs) unexpectedly enhances, rather than diminishes, delivery efficacy and cytotoxicity to human cancer cells. This highly unexpected enhancement occurs even when the PEG-chains coating the NP are in the transition regime between the mushroom and brush conformations. Cryogenic transmission electron microscopy (TEM) of PEG-CLPTX NPs shows that PEG causes the proliferation of a mixture of sterically stabilized nanometer-scale vesicles and anisotropic micelles (e.g., bicelles). Remarkably, the onset of bicelles at sub-monolayer concentrations of the PEG coat has to our knowledge not been previously reported; it was previously thought that PEG-lipid in this composition regime was incorporated into vesicles but did not alter their shape. Confocal microscopy and flow cytometry reveal significantly greater PTX cell uptake from stabilized PEG-CLPTX NPs (vesicles and bicelles) in contrast to bare CLPTX NPs, which can aggregate in cell medium. This underscores the ability of steric stabilization to facilitate NP entry into cells via distinct size-dependent endocytic pathways, some of which cannot transport large NP aggregates into cells. This study highlights the value of understanding how PEGylation alters NP shape and structure, and thus NP efficacy, to design next-generation stealth drug carriers that integrate active cell-targeting strategies into NPs for in vivo delivery.

Engineering the PP7 Virus Capsid as a Peptide Display Platform.

As self-assembling polyvalent nanoscale structures that can tolerate substantial genetic and chemical modification, virus-like particles are useful in a variety of fields. Here we describe the genetic modification and structural characterization of the Leviviridae PP7 capsid protein as a platform for the presentation of functional polypeptides. This particle was shown to tolerate the display of sequences from 1 kDa (a cell penetrating peptide) to 14 kDa (the Fc-binding double Z-domain) on its exterior surface as C-terminal genetic fusions to the coat protein. In addition, a dimeric construct allowed the presentation of exogenous loops between capsid monomers and the simultaneous presentation of two different peptides at different positions on the icosahedral structure. The PP7 particle is thereby significantly more tolerant of these types of polypeptide additions than Qß and MS2, the other Leviviridae-derived VLPs in common use.

Structure-based design of a quadrivalent fusion glycoprotein vaccine for human parainfluenza virus types 1-4.

Parainfluenza virus types 1-4 (PIV1-4) are highly infectious human pathogens, of which PIV3 is most commonly responsible for severe respiratory illness in newborns, elderly, and immunocompromised individuals. To obtain a vaccine effective against all four PIV types, we engineered mutations in each of the four PIV fusion (F) glycoproteins to stabilize their metastable prefusion states, as such stabilization had previously enabled the elicitation of high-titer neutralizing antibodies against the related respiratory syncytial virus. A cryoelectron microscopy structure of an engineered PIV3 F prefusion-stabilized trimer, bound to the prefusion-specific antibody PIA174, revealed atomic-level details for how introduced mutations improved stability as well as how a single PIA174 antibody recognized the trimeric apex of prefusion PIV3 F. Nine combinations of six newly identified disulfides and two cavity-filling mutations stabilized the prefusion PIV3 F immunogens and induced 200- to 500-fold higher neutralizing titers in mice than were elicited by PIV3 F in the postfusion conformation. For PIV1, PIV2, and PIV4, we also obtained stabilized prefusion Fs, for which prefusion versus postfusion titers were 2- to 20-fold higher. Elicited murine responses were PIV type-specific, with little cross-neutralization of other PIVs. In nonhuman primates (NHPs), quadrivalent immunization with prefusion-stabilized Fs from PIV1-4 consistently induced potent neutralizing responses against all four PIVs. For PIV3, the average elicited NHP titer from the quadrivalent immunization was more than fivefold higher than any titer observed in a cohort of over 100 human adults, highlighting the ability of a prefusion-stabilized immunogen to elicit especially potent neutralization.

Structure of the insulin receptor-insulin complex by single-particle cryo-EM analysis.

The insulin receptor is a dimeric protein that has a crucial role in controlling glucose homeostasis, regulating lipid, protein and carbohydrate metabolism, and modulating brain neurotransmitter levels. Insulin receptor dysfunction has been associated with many diseases, including diabetes, cancer and Alzheimer's disease. The primary sequence of the receptor has been known since the 1980s, and is composed of an extracellular portion (the ectodomain, ECD), a single transmembrane helix and an intracellular tyrosine kinase domain. Binding of insulin to the dimeric ECD triggers auto-phosphorylation of the tyrosine kinase domain and subsequent activation of downstream signalling molecules. Biochemical and mutagenesis data have identified two putative insulin-binding sites, S1 and S2. The structures of insulin bound to an ECD fragment containing S1 and of the apo ectodomain have previously been reported, but details of insulin binding to the full receptor and the signal propagation mechanism are still not understood. Here we report single-particle cryo-electron microscopy reconstructions of the 1:2 (4.3 Å) and 1:1 (7.4 Å) complexes of the insulin receptor ECD dimer with insulin. The symmetrical 4.3 Å structure shows two insulin molecules per dimer, each bound between the leucine-rich subdomain L1 of one monomer and the first fibronectin-like domain (FnIII-1) of the other monomer, and making extensive interactions with the α-subunit C-terminal helix (α-CT helix). The 7.4 Å structure has only one similarly bound insulin per receptor dimer. The structures confirm the binding interactions at S1 and define the full S2 binding site. These insulin receptor states suggest that recruitment of the α-CT helix upon binding of the first insulin changes the relative subdomain orientations and triggers downstream signal propagation.

Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex.

All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes.

Characterization of virus-like particles in GARDASIL® by cryo transmission electron microscopy.

Cryo-transmission electron microscopy (cryoTEM) is a powerful characterization method for assessing the structural properties of biopharmaceutical nanoparticles, including Virus Like Particle-based vaccines. We demonstrate the method using the Human Papilloma Virus (HPV) VLPs in GARDASIL®. CryoTEM, coupled to automated data collection and analysis, was used to acquire images of the particles in their hydrated state, determine their morphological characteristics, and confirm the integrity of the particles when absorbed to aluminum adjuvant. In addition, we determined the three-dimensional structure of the VLPs, both alone and when interacting with neutralizing antibodies. Two modes of binding of two different neutralizing antibodies were apparent; for HPV type 11 saturated with H11.B2, 72 potential Fab binding sites were observed at the center of each capsomer, whereas for HPV 16 interacting with H16.V5, it appears that 60 pentamers (each neighboring 6 other pentamers) bind five Fabs per pentamer, for the total of 300 potential Fab binding sites per VLP.

Maximizing the potential of electron cryomicroscopy data collected using direct detectors.

Single-particle electron cryomicroscopy is undergoing a technical revolution due to the recent developments of direct detectors. These new recording devices detect electrons directly (i.e. without conversion into light) and feature significantly improved detective quantum efficiencies and readout rates as compared to photographic films or CCDs. We evaluated here the potential of one such detector (Gatan K2 Summit) to enable the achievement of near-atomic resolution reconstructions of biological specimens when coupled to a widely used, mid-range transmission electron microscope (FEI TF20 Twin). Compensating for beam-induced motion and stage drift provided a 4.4Å resolution map of Sulfolobus turreted icosahedral virus (STIV), which we used as a test particle in this study. Several motion correction and dose fractionation procedures were explored and we describe their influence on the resolution of the final reconstruction. We also compared the quality of this data to that collected with a FEI Titan Krios microscope equipped with a Falcon I direct detector, which provides a benchmark for data collected using a high-end electron microscope.

Engineered nanostructured ß-sheet peptides protect membrane proteins.

We designed ß-strand peptides that stabilize integral membrane proteins (IMPs). ß-strand peptides self-assemble in solution as filaments and become restructured upon association with IMPs; resulting IMP-ß-strand peptide complexes resisted aggregation when diluted in detergent-free buffer and were visible as stable, single particles with low detergent background in electron micrographs. ß-strand peptides enabled clear visualization of flexible conformations in the highly dynamic ATP-binding cassette (ABC) transporter MsbA.

Single-particle EM reveals extensive conformational variability of the Ltn1 E3 ligase.

Ltn1 is a 180-kDa E3 ubiquitin ligase that associates with ribosomes and marks certain aberrant, translationally arrested nascent polypeptide chains for proteasomal degradation. In addition to its evolutionarily conserved large size, Ltn1 is characterized by the presence of a conserved N terminus, HEAT/ARM repeats predicted to comprise the majority of the protein, and a C-terminal catalytic RING domain, although the protein's exact structure is unknown. We used numerous single-particle EM strategies to characterize Ltn1's structure based on negative stain and vitreous ice data. Two-dimensional classifications and subsequent 3D reconstructions of electron density maps show that Ltn1 has an elongated form and presents a continuum of conformational states about two flexible hinge regions, whereas its overall architecture is reminiscent of multisubunit cullin-RING ubiquitin ligase complexes. We propose a model of Ltn1 function based on its conformational variability and flexibility that describes how these features may play a role in cotranslational protein quality control.

Organization of the influenza virus replication machinery.

Influenza virus ribonucleoprotein complexes (RNPs) are central to the viral life cycle and in adaptation to new host species. RNPs are composed of the viral genome, viral polymerase, and many copies of the viral nucleoprotein. In vitro cell expression of all RNP protein components with four of the eight influenza virus gene segments enabled structural determination of native influenza virus RNPs by means of cryogenic electron microscopy (cryo-EM). The cryo-EM structure reveals the architecture and organization of the native RNP, defining the attributes of its largely helical structure and how polymerase interacts with nucleoprotein and the viral genome. Observations of branched-RNP structures in negative-stain electron microscopy and their putative identification as replication intermediates suggest a mechanism for viral replication by a second polymerase on the RNP template.

Disassembly and reassembly of human papillomavirus virus-like particles produces more virion-like antibody reactivity.

BACKGROUND: Human papillomavirus (HPV) vaccines based on major capsid protein L1 are licensed in over 100 countries to prevent HPV infections. The yeast-derived recombinant quadrivalent HPV L1 vaccine, GARDASIL(R), has played an important role in reducing cancer and genital warts since its introduction in 2006. The L1 proteins self-assemble into virus-like particles (VLPs). RESULTS: VLPs were subjected to post-purification disassembly and reassembly (D/R) treatment during bioprocessing to improve VLP immunoreactivity and stability. The post-D/R HPV16 VLPs and their complex with H16.V5 neutralizing antibody Fab fragments were visualized by cryo electron microscopy, showing VLPs densely decorated with antibody. Along with structural improvements, post-D/R VLPs showed markedly higher antigenicity to conformational and neutralizing monoclonal antibodies (mAbs) H16.V5, H16.E70 and H263.A2, whereas binding to mAbs recognizing linear epitopes (H16.J4, H16.O7, and H16.H5) was greatly reduced. Strikingly, post-D/R VLPs showed no detectable binding to H16.H5, indicating that the H16.H5 epitope is not accessible in fully assembled VLPs. An atomic homology model of the entire HPV16 VLP was generated based on previously determined high-resolution structures of bovine papillomavirus and HPV16 L1 pentameric capsomeres. CONCLUSIONS: D/R treatment of HPV16 L1 VLPs produces more homogeneous VLPs with more virion-like antibody reactivity. These effects can be attributed to a combination of more complete and regular assembly of the VLPs, better folding of L1, reduced non-specific disulfide-mediated aggregation and increased stability of the VLPs. Markedly different antigenicity of HPV16 VLPs was observed upon D/R treatment with a panel of monoclonal antibodies targeting neutralization sensitive epitopes. Multiple epitope-specific assays with a panel of mAbs with different properties and epitopes are required to gain a better understanding of the immunochemical properties of VLPs and to correlate the observed changes at the molecular level. Mapping of known antibody epitopes to the homology model explains the changes in antibody reactivity upon D/R. In particular, the H16.H5 epitope is partially occluded by intercapsomeric interactions involving the L1 C-terminal arm. The homology model allows a more precise mapping of antibody epitopes. This work provides a better understanding of VLPs in current vaccines and could guide the design of improved vaccines or therapeutics.

Nucleotide-dependent conformational changes in the N-Ethylmaleimide Sensitive Factor (NSF) and their potential role in SNARE complex disassembly.

Homohexameric, N-Ethylmaleimide Sensitive Factor (NSF) disassembles Soluble NSF Attachment Protein Receptor (SNARE) complexes after membrane fusion, an essential step in vesicular trafficking. NSF contains three domains (NSF-N, NSF-D1, and NSF-D2), each contributing to activity. We combined electron microscopic (EM) analysis, analytical ultracentrifugation (AU) and functional mutagenesis to visualize NSF's ATPase cycle. 3D density maps show that NSF-D2 remains stable, whereas NSF-N undergoes large conformational changes. NSF-Ns splay out perpendicular to the ADP-bound hexamer and twist upwards upon ATP binding, producing a more compact structure. These conformations were confirmed by hydrodynamic, AU measurements: NSF-ATP sediments faster with a lower frictional ratio (f/f(0)). Hydrodynamic analyses of NSF mutants, with specific functional defects, define the structures underlying these conformational changes. Mapping mutations onto our 3D models allows interpretation of the domain movement and suggests a mechanism for NSF binding to and disassembly of SNARE complexes.

A new model for binding of kinesin 13 to curved microtubule protofilaments.

Kinesin motor proteins use adenosine triphosphate hydrolysis to do work on microtubules (MTs). Most kinesins walk along the MT, but class 13 kinesins instead uniquely recognize MT ends and depolymerize MT protofilaments. We have used electron microscopy (EM) to understand the molecular interactions by which kinesin 13 performs these tasks. Although a construct of only the motor domain of kinesin 13 binds to every heterodimer of a tubulin ring, a construct containing the neck and the motor domain occupies alternate binding sites. Likewise, EM maps of the dimeric full-length (FL) protein exhibit alternate site binding but reveal density for only one of two motor heads. These results indicate that the second head of dimeric kinesin 13 does not have access to adjacent binding sites on the curved protofilament and suggest that the neck alone is sufficient to obstruct access. Additionally, the FL construct promotes increased stacking of rings compared with other constructs. Together, these data suggest a model for kinesin 13 depolymerization in which increased efficiency is achieved by binding of one kinesin 13 molecule to adjacent protofilaments.

Nucleotide dependent packing differences in helical crystals of the ABC transporter MsbA.

Bacterial ATP binding cassette (ABC) exporters fulfill a wide variety of transmembrane transport roles and are homologous to the human multidrug resistance P-glycoprotein. Recent X-ray structures of the exporters MsbA and Sav1866 have begun to describe the conformational changes that accompany the ABC transport cycle. Here we present cryo-electron microscopy structures of MsbA reconstituted into a lipid bilayer. Using ATPase inhibitors, we captured three nucleotide transition states of the transporter that were subsequently reconstituted into helical arrays. The enzyme-substrate complex (trapped by ADP-aluminum fluoride or AMPPNP) crystallized in a different helical lattice than the enzyme-product complex (trapped by ADP-vanadate). Approximately 20A resolution maps were calculated for each state and revealed MsbA to be a dimer with a large channel between the membrane spanning domains, similar to the outward facing crystal structures of MsbA and Sav1866. This suggests that while there are likely structural differences between the nucleotide transition states, membrane embedded MsbA remains in an outward facing conformation while nucleotide is bound.

Rapid routine structure determination of macromolecular assemblies using electron microscopy: current progress and further challenges.

Although the methodology of molecular microscopy has enormous potential, it is time consuming and labor intensive. The techniques required to produce a three-dimensional (3D) electron density map of a macromolecular structure normally require manual operation of an electron microscope by a skilled operator and manual supervision of the sometimes complex software needed for analysis and calculation of 3D maps. Systems to automate the process of data acquisition from an electron microscope are being developing and these systems are being integrated with specimen handling operations and post acquisition data processing. Here, the current performance of our existing systems and the future challenges involved in substantially improving both the sustained throughput and the yield of automated data collection and analysis are reported.

Windex: a toolset for indexing helices.

We describe here a set of procedures and algorithms that may be used as an aid in determining the indexing rule of a helical specimen. Crystallizing macromolecules into helical arrays has the potential to speed up and simplify the process of three-dimensional reconstruction of the macromolecular structure. The process of helical reconstruction has been largely automated except for the critical first step of indexing the helical diffraction pattern. This is quite often the rate-limiting step in the overall process, particularly in the case of large helical tubes, which have complicated helical diffraction patterns that may vary from tube to tube. We have developed a set of procedures, supported by a graphical user interface, that provide a straightforward and semi-automated approach to indexing a helical structure. The new procedures have been tested using a number of helical specimens, including TMV, acto-myosin, decorated microtubules, and a variety of helical tubes of a bacterial membrane protein.

Distinct conformations of the kinesin Unc104 neck regulate a monomer to dimer motor transition.

Caenhorhabditis elegans Unc104 kinesin transports synaptic vesicles at rapid velocities. Unc104 is primarily monomeric in solution, but recent motility studies suggest that it may dimerize when concentrated on membranes. Using cryo-electron microscopy, we observe two conformations of microtubule-bound Unc104: a monomeric state in which the two neck helices form an intramolecular, parallel coiled coil; and a dimeric state in which the neck helices form an intermolecular coiled coil. The intramolecular folded conformation is abolished by deletion of a flexible hinge separating the neck helices, indicating that it acts as a spacer to accommodate the parallel coiled-coil configuration. The neck hinge deletion mutation does not alter motor velocity in vitro but produces a severe uncoordinated phenotype in transgenic C. elegans, suggesting that the folded conformation plays an important role in motor regulation. We suggest that the Unc104 neck regulates motility by switching from a self-folded, repressed state to a dimerized conformation that can support fast processive movement.