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Integrative Structural Biology and Its Moment in the Spotlight

Team TFS
Team TFS
covidIf we needed evidence that virus research is an important area to focus on, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic and cumulative COVID-19 human health impact have provided a global case study for the need to maintain a responsive and effective ability to rapidly understand emerging viruses.

Currently, structural biology is having its moment in the sun, playing a crucial role in the research community’s response to the pandemic. It has enabled the characterization of the SARS-CoV-2 virus structure by providing crucial information on the virus spike glycoprotein, receptor-binding domain (RBD), virus-host cell interaction, vaccine development,  drug candidates identification and immune response comprehension. The primary driver of these studies has been the use of cryogenic electron microscopy (cryo-EM). Cryo-EM enables researchers to look at protein structures and protein complexes at near atomic resolution. With cryo-EM, structural biologists can not only study viruses in their biologically relevant forms, but they can also determine how these samples interact with other marker molecules or compounds. This information can help provide detailed insights into virus structure and function, which might enable virus detection and inform drug and vaccine programs designed to prevent infection and disease.

However, solving the structure of large dynamic complexes often requires integrating several complementary techniques to cryo-EM such as mass spectrometry (MS). Technology developments in MS have given rise to several applications to structural biology, both at the single protein and protein complex level. The primary advantages of MS-based techniques are the ability to perform experiments at proteome scale, to analyze proteins in their native biological state, and to reduce the minimum required sample size.

MS offers many approaches to gain insights into viral structure and behavior. As it pertains to the current pandemic, MS based glycoproteomics have been used to map recombinant spike glycoproteins of the SARS-CoV-2 virus, including sites of glycosylation and glycan compositions. Similar approaches have been used to examine the actual SARS-CoV-2 virus spike protein, including the host receptor that binds to the spike protein.

Another MS approach, called Native MS, has been used to examine the viral protease, known as the main protease (Mpro). This is an important protease that is involved in viral gene expression and replication, and the ideal drug targets for antiviral activity. Native MS has been used to develop a binding assay for SARS-CoV-2 Mpro to different protease inhibitors such as Ebselen, Disulfiram, Carmofur, PX-12, Tideglusib, Shikonin, Boceprevir, GC-376, calpain inhibitors II, XII, AWJ246, 247, and 248. It also has been used to characterize the functional unit of Mpro and look at the assembly state and RNA binding properties of the full-length nucleocapsid protein. 

Crosslinking MS (XL-MS) is another powerful structural tool in the MS toolbox. Ultraviolet (UV) crosslinking to capture the interaction between SARS-CoV-2 RNA and proteins has enabled researchers to examine a quantitative picture of the human proteome that directly binds the SARS-CoV-2 RNA in infec.... It also has allowed researchers to look at potential therapeutic treatment for COVID-19. For example, researchers have used XL-MS to identify highly potent neutralizing nanobodies (Nbs) generated from llamas that bind to the SARS-CoV-2 spike protein RBD. These are a low-cost alternative to traditional monoclonal antibodies that inhibit viral infection.

Researchers at University of California San Francisco have used another MS approach called affinity-purification mass spectrometry (AP-MS) to identify protein-protein interactions between SARS-CoV-2 proteins and human host cell proteins. This has enabled the group to create a map of proteins involved in viral infection and replication, making it possible to see which of the existing approved drugs might be repurposed to deal with a SARS-CoV-2 infection by targeting the proteins identified in their protein-protein interaction map.

One of the more commonly used MS tools for structural biology has been hydrogen deuterium exchange mass spectrometry (HDX-MS). HDX-MS analysis can be used to obtain information on structure, protein-protein or protein-ligand interaction sites, allosteric effects, intrinsic disorder, and conformational changes induced by posttranslational modifications (PTMs). In a study by Hansen et al., where they examined a large panel of antibodies against the  the SARS-CoV-2 spike protein, they were able to filter out several powerful neutralizing antibodies. In order to examine the binding regions of their mAbs on the spike proteins RBD, they employed HDX-MS to reveal information about the regions of antibody contact to the spike protein’s RBD and compared that with how the host ACE2 protein might interact with spike protein RBD.

Whether the focus is on studying the virus itself, virus-host interactions, host immunity, the immune system, or virus cellular machinery, proteins play crucial roles. The host proteome will go through a number of processes including production, degradation and spatial reorganization. Understanding these processes will enable researchers to develop strategies to better tackle the virus under study. The vast majority of these studies involve looking at very complex structures involved in a myriad of interactions. Thus, the study of viruses has benefitted tremendously from the rapid advancement of structural biology tools including MS. The integration of MS tools such as HDX-MS, XL-MS, AP-MS, native MS, and PTM characterization has enabled the study of these complexes and the interactions that they participate in, which promotes a better understanding of protein function and mechanism of action in biological systems. Furthermore, MS tools are complementary to traditional techniques such as cryo-EM, X-ray crystallography and NMR by either filling in or extending information that these techniques miss.

To find out more about virus research visit the Viral Proteomics & Metabolomics Mass Spectrometry page.
1 Comment
Team TFS
Team TFS
Thank you Andreas! Your blog is very interesting