Joost Snijder

Probing the biophysical interplay between a viral genome and its capsid

The interaction between a viral capsid and its genome governs crucial steps in the life cycle of a virus, such as assembly and genome uncoating. Tuning cargo-capsid interactions is also essential for successful design and cargo delivery in engineered viral systems. Here we investigate the interplay between cargo and capsid for the picorna-like Triatoma virus using a combined native mass spectrometry and atomic force microscopy approach. We propose a topology and assembly model in which heterotrimeric pentons that consist of five copies of structural proteins VP1, VP2 and VP3 are the free principal units of assembly. The interpenton contacts are established primarily by VP2. The dual role of the genome is first to stabilize the densely packed virion and, second, on an increase in pH to trigger uncoating by relaxing the stabilizing interactions with the capsid. Uncoating occurs through a labile intermediate state of the virion that reversibly disassembles into pentons with the concomitant release of protein VP4.

Defining the Stoichiometry and Cargo Load of Viral and Bacterial Nanoparticles by Orbitrap Mass Spectrometry

Accurate mass analysis can provide useful information on the stoichiometry and composition of protein-based particles, such as virus-like assemblies. For applications in nanotechnology and medicine, such nanoparticles are loaded with foreign cargos, making accurate mass information essential to define the cargo load. Here, we describe modifications to an Orbitrap mass spectrometer that enable high mass analysis of several virus-like nanoparticles up to 4.5 MDa in mass. This allows the accurate determination of the composition of virus-like particles. The modified instrument is utilized to determine the cargo load of bacterial encapsulin nanoparticles that were engineered to encapsulate foreign cargo proteins. We find that encapsulin packages from 8 up to 12 cargo proteins, thereby quantifying cargo load but also showing the ensemble spread. In addition, we determined the previously unknown stoichiometry of the three different splice variants of the capsid protein in adeno-associated virus (AAV) capsids, showing that symmetry is broken and assembly is heterogeneous and stochastic. These results demonstrate the potential of high-resolution mass analysis of protein-based nanoparticles, with widespread applications in chemical biology and nanotechnology.

Analytical Approaches for Size and Mass Analysis of Large Protein Assemblies

Analysis of the size and mass of nanoparticles, whether they are natural biomacromolecular or synthetic supramolecular assemblies, is an important step in the characterization of such molecular species. In recent years, electrospray ionization (ESI) has emerged as a technology through which particles with masses up to 100 MDa can be ionized and transferred into the gas phase, preparing them for accurate mass analysis. Here we review currently used methodologies, with a clear focus on native mass spectrometry (MS). Additional complementary methodologies are also covered, including ion-mobility analysis, nanomechanical mass sensors, and charge-detection MS. The literature discussed clearly demonstrates the great potential of ESI-based methodologies for the size and mass analysis of nanoparticles, including very large naturally occurring protein assemblies. The analytical approaches discussed are powerful tools in not only structural biology, but also nanotechnology.

Boundaries of Mass Resolution in Native Mass Spectrometry

AbstractOver the last two decades, native mass spectrometry (MS) has emerged as a valuable tool to study intact proteins and noncovalent protein complexes. Studied experimental systems range from small-molecule (drug)–protein interactions, to nanomachineries such as the proteasome and ribosome, to even virus assembly. In native MS, ions attain high m/z values, requiring special mass analyzers for their detection. Depending on the particular mass analyzer used, instrumental mass resolution does often decrease at higher m/z but can still be above a couple of thousand at m/z 5000. However, the mass resolving power obtained on charge states of protein complexes in this m/z region is experimentally found to remain well below the inherent instrument resolution of the mass analyzers employed. Here, we inquire into reasons for this discrepancy and ask how native MS would benefit from higher instrumental mass resolution. To answer this question, we discuss advantages and shortcomings of mass analyzers used to study intact biomolecules and biomolecular complexes in their native state, and we review which other factors determine mass resolving power in native MS analyses. Recent examples from the literature are given to illustrate the current status and limitations. Figureᅟ

Integrin and Defensin Modulate the Mechanical Properties of Adenovirus

ABSTRACT The propensity for capsid disassembly and uncoating of human adenovirus is modulated by interactions with host cell molecules like integrins and alpha defensins. Here, we use atomic force microscopy (AFM) nanoindentation to elucidate, at the single-particle level, the mechanism by which binding of these host molecules affects virus particle elasticity. Our results demonstrate the direct link between integrin or defensin binding and the mechanical properties of the virus. We show that the structure and geometry of adenovirus result in an anisotropic elastic response that relates to icosahedral symmetry. This elastic response changes upon binding host molecules. Whereas integrin binding softens the vertex regions, binding of a human alpha defensin has exactly the opposite effect. Our results reveal that the ability of these host molecules to influence adenovirus disassembly correlates with a direct effect on the elastic strength of the penton region. Host factors that influence adenovirus infectivity thus modulate the elastic properties of the capsid. Our findings reveal a direct link between virus-host interactions and capsid mechanics.

Adenovirus composition, proteolysis, and disassembly studied by in-depth qualitative and quantitative proteomics.

Background: Adenoviruses (AdV) are broadly employed as gene delivery vectors. Results: Copy numbers of all AdV proteins were measured, and the release of proteins upon heat stress investigated. Conclusion: The viral protease plays a distinct role in the segmented release of AdV proteins. Significance: Our characterization by mass spectrometry provides new insight in HAdV disassembly during entry into host cells. Using high-resolution MS-based proteomics in combination with multiple protease digestion, we profiled, with on average 90% sequence coverage, all 13 viral proteins present in an human adenovirus (HAdV) vector. This in-depth profile provided multiple peptide-based evidence on intrinsic protease activity affecting several HAdV proteins. Next, the generated peptide library was used to develop a targeted proteomics method using selected reaction monitoring (SRM) aimed at quantitative profiling of the stoichiometry of all 13 proteins present in the HAdV. We also used this method to probe the release of specific virus proteins initiated by thermal stimulation, mimicking the early stage of HAdV disassembly during entry into host cells. We confirmed the copy numbers of the most well characterized viral capsid components and established the copy numbers for proteins whose stoichiometry has so far not been accurately defined. We also found that heating HAdV induces the complete release of the penton base and fiber proteins as well as a substantial release of protein VIII and VI. For these latter proteins, maturational proteolysis by the adenoviral protease leads to the differential release of fragments with certain peptides being fully released and others largely retained in the AdV particles. This information is likely to be beneficial for the ongoing interpretation of high resolution cryoEM and x-ray electron density maps.

Glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy.

The threat of a major coronavirus pandemic urges the development of strategies to combat these pathogens. Human coronavirus NL63 (HCoV-NL63) is an α-coronavirus that can cause severe lower-respiratory-tract infections requiring hospitalization. We report here the 3.4-Å-resolution cryo-EM reconstruction of the HCoV-NL63 coronavirus spike glycoprotein trimer, which mediates entry into host cells and is the main target of neutralizing antibodies during infection. The map resolves the extensive glycan shield obstructing the protein surface and, in combination with mass spectrometry, provides a structural framework to understand the accessibility to antibodies. The structure reveals the complete architecture of the fusion machinery including the triggering loop and the C-terminal domains, which contribute to anchoring the trimer to the viral membrane. Our data further suggest that HCoV-NL63 and other coronaviruses use molecular trickery, based on epitope masking with glycans and activating conformational changes, to evade the immune system of infected hosts.

Self-sorting of foreign proteins in a bacterial nanocompartment

Nature uses bottom-up approaches for the controlled assembly of highly ordered hierarchical structures with defined functionality, such as organelles, molecular motors, and transmembrane pumps. The field of bionanotechnology draws inspiration from nature by utilizing biomolecular building blocks such as DNA, proteins, and lipids, for the (self-) assembly of new structures for applications in biomedicine, optics, or electronics. Among the toolbox of available building blocks, proteins that form cage-like structures, such as viruses and virus-like particles, have been of particular interest since they form highly symmetrical assemblies and can be readily modified genetically or chemically both on the outer or inner surface. Bacterial encapsulins are a class of nonviral protein cages that self-assemble in vivo into stable icosahedral structures. Using teal fluorescent proteins (TFP) engineered with a specific native C-terminal docking sequence, we report the molecular self-sorting and selective packaging of TFP cargo into bacterial encapsulins during in vivo assembly. Using native mass spectrometry techniques, we show that loading of either monomeric or dimeric TFP cargo occurs with unprecedented high fidelity and exceptional loading accuracy. Such self-assembling systems equipped with self-sorting capabilities would open up exciting opportunities in nanotechnology, for example, as artificial (molecular storage or detoxification) organelles or as artificial cell factories for in situ biocatalysis.

Insight into cyanobacterial circadian timing from structural details of the KaiB–KaiC interaction

Significance The Kai system is a widely studied model in theoretical biology and systems biology. It is to date the only known circadian clock that can be reconstituted in vitro. Essential to the rhythmicity of the system is the formation of the KaiC–KaiB complex. Many aspects of this interaction, such as the mode of binding of KaiB, the stoichiometry of the interaction, and the exact binding interfaces have long remained ambiguous. We present a mass spectrometry-based structural model of the KaiC–KaiB interaction that answers many of these outstanding questions on the basis of direct experimental evidence. This structural model sheds light on the intricate workings of the in vitro oscillator. Circadian timing in cyanobacteria is determined by the Kai system consisting of KaiA, KaiB, and KaiC. Interactions between Kai proteins change the phosphorylation status of KaiC, defining the phase of circadian timing. The KaiC–KaiB interaction is crucial for the circadian rhythm to enter the dephosphorylation phase but it is not well understood. Using mass spectrometry to characterize Kai complexes, we found that KaiB forms monomers, dimers, and tetramers. The monomer is the unit that interacts with KaiC, with six KaiB monomers binding to one KaiC hexamer. Hydrogen–deuterium exchange MS reveals structural changes in KaiC upon binding of KaiB in both the CI and CII domains, showing allosteric coupling upon KaiB binding. Based on this information we propose a model of the KaiB–KaiC complex and hypothesize that the allosteric changes observed upon complex formation relate to coupling KaiC ATPase activity with KaiB binding and to sequestration of KaiA dimers into KaiCBA complexes.

Self-assembly and characterization of small and monodisperse dye nanospheres in a protein cage

Phthalocyanines (Pc) are dyes in widespread use in materials science and nanotechnology, with numerous applications in medicine, photonics, electronics and energy conversion. With the aim to construct biohybrid materials, we here prepared and analyzed the structure of two Pc-loaded virus-like particles (VLP) with diameters of 20 and 28 nm (i.e., T = 1 and T = 3 icosahedral symmetries, respectively). Our cryo-electron microscopy (cryo-EM) studies show an unprecedented, very high level of Pc molecule organization within both VLP. We found that 10 nm diameter nanospheres form inside the T = 1 VLP by self-assembly of supramolecular Pc stacks. Monodisperse, self-assembled organic dye nanospheres were not previously known, and are a consequence of capsid-imposed symmetry and size constraints. The Pc cargo also produces major changes in the protein cage structure and in the mechanical properties of the VLP. Pc-loaded VLP are potential photosensitizer/carrier systems in photodynamic therapy (PDT), for which their mechanical behaviour must be characterized. Many optoelectronic applications of Pc dyes, on the other hand, are dependent on dye organization at the nanoscale level. Our multidisciplinary study thus opens the way towards nanomedical and nanotechnological uses of these functional molecules.