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High-field NMR and global collaboration drive Covid-19 protein research at Göttingen

As part of a 50-strong international collaboration of NMR experts, a team led by Prof. Dr. Markus Zweckstetter at the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany, has been uncovering insights into the hidden workings of Covid-19. Using one of only a few 1.2 GHz NMR instruments in the world, his team is helping to elucidate the structure and interactions of the nucleocapsid protein within the SARS-CoV-2 virus and in doing so, identify promising options for drug targets.

Research in a new dimension

Physics and biology are often viewed as being at the opposite extremes of the scientific spectrum, and many scientists tend to follow one route or the other. But for those prepared to bridge the divide, there are great rewards, and Prof. Dr. Markus Zweckstetter is one of those people.

Prof. Zweckstetter originally trained in physics, but he quickly turned his skills to biophysical chemistry, completing his Ph.D. in biomolecular nuclear magnetic resonance (NMR) spectroscopy at the Max Planck Institute for Biochemistry in Martinsried, Germany, in 1998. This was followed by postdoctoral studies at the same institution, and at the National Institutes of Health in Bethesda, USA. He then moved to Göttingen, Germany, where since 2001 he has headed a group at the Max Planck Institute for Biophysical Chemistry, and from 2012 also at the German Center for Neurodegenerative Diseases (DZNE). Prof. Zweckstetter received two European Research Council (ERC) grants: an ERC consolidator phase in 2011 and an ERC Advanced Grant in 2018, which help him, and his team uncover protein structure and function using the power of NMR spectroscopy.

Zooming into biomolecules

Throughout his time at Göttingen, Prof. Zweckstetter has been a keen advocate of NMR, and particularly for its ability to shed light on the conformations of biomolecules and the modes of action of small molecules with potential use as drugs.

As he says: “I’ve always been interested in molecular processes as well as physics, and very early on in my career I saw that NMR offered the opportunity to combine insights from both fields. Proteins, with their unparalleled diversity and complexity, offer an intriguing and fertile testing ground for NMR technology, and one that for well over 25 years has inspired me in my research.”

Prof. Zweckstetter explains that NMR is a uniquely powerful method to interrogate biomolecules at high resolution: “Because it allows you to study proteins, oligonucleotides and RNA under physiological conditions – and even inside cells – it is a perfect method for working out molecular structures and interactions in detail, and so to develop potential drugs”.

A prominent example of his work was determining the high-resolution structure of the mammalian translocator protein (TSPO), which has increased expression in areas of brain injury and inflammation. The research, which was published in the journal Science in 2014,[1] demonstrated that coupling TSPO to a diagnostic ligand called PK11195 stabilized its structure, allowing the structure to be determined as a bundle of five helices. NMR was crucial to this work, by allowing the proximity of specific 1H, 13C and 15N nuclei to be determined using nuclear Overhauser spectroscopy (NOESY). The result was a much better understanding of how TSPO recognizes and binds to diagnostic markers and drugs, with clear diagnostic and therapeutic implications.

Most of Prof. Zweckstetter’s research has been directly related to the proteins involved in neurodegenerative disorders, with special focus on three peptides – tau, α-synuclein, and amyloid-β – that form insoluble deposits that are hallmarks of Alzheimer’s disease and Parkinson’s disease. For example, in recent work looking at the processes that underlie the formation of insoluble deposits of tau in the brain of patients with Alzheimer’s disease, Prof. Zweckstetter’s team identified liquid-liquid phase separation as a critical driving factor [2]. The molecular crowding of tau that is a consequence of the phase separation process was uncovered by two-dimensional 1H/15N correlation spectroscopy (HSQC) of tau coupled to a paramagnetic tag. The findings in this study triggered a wide range of research in different labs worldwide.
 

Prof. Dr. Markus Zweckstetter is head of the research group on “Structure determination of proteins using NMR” at the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany. Since 2012, he has also led the “Translational Structural Biology” group at the German Center for Neurodegenerative Diseases (DZNE), at the University Medical Center in Göttingen.

Understanding the nucleocapsid protein in SARS-CoV-2

Collaboration has always played a key part in Prof. Zweckstetter’s research, but this aspect has been taken to a new level in recent months, with his involvement in the Covid-19 NMR consortium: an international collaboration of NMR experts, that aims to determine the RNA structure of SARS-CoV-2 and its proteins using NMR spectroscopy, and in doing so provide insights into their ‘drugability’ by small molecules.[3]

This consortium was initiated by the publication of a paper by Prof. Zweckstetter in late 2020,[4] which described the discovery that a protein within SARS-CoV-2 (the causative virus behind the Covid-19 pandemic) forms microdroplets with the RNA of the virus. To uncover this, the team combined molecular dynamics simulations with NMR spectroscopy of 1H, 13C and 15N nuclei, using NOESY, HSQC and total correlation spectroscopy (TOCSY), with an example of the sorts of insights obtained shown in Figure 2.

This publication attracted the attention of the consortium’s leader, Prof. Dr. Harald Schwalbe at the Goethe-Universität in Frankfurt, Germany, who subsequently invited Prof. Zweckstetter to join the group. He readily accepted, both in order to benefit from the Schwalbe group’s expertise in RNA, and also because collaborating would give his team access to samples of SARS-CoV-2 viral RNA. In subsequent months, this enabled them to set up experiments to study the interplay between the RNA and the above-mentioned protein, known as the nucleocapsid protein or ‘N protein’.

Prof. Zweckstetter explains that many early studies of SARS-CoV-2 focused on the so-called ‘spike protein’, because this is involved in communication with the host cell. But as understanding of SARS-CoV-2 has evolved, it has become apparent that N protein also has a key role. This is because, as he says, “It not only protects the RNA from degradation, but enables the transcription machinery to cluster, and so enhances its ability to replicate”.

The upshot of this, says Prof. Zweckstetter, is that many groups around the world are now studying N protein, with a view to assessing it as a target for treatment of Covid-19. For example, by interfering with the formation of N protein microdroplets, the viral RNA might become more vulnerable to external damage and less able to replicate reliably. He adds that another aspect of the work on N protein is to investigate the involvement of kinases in phosphorylating certain residues of N protein, as these enzymes could be promising targets for small-molecule inhibitors.
 

Figure 1: Molecular crowding of the Alzheimer’s disease-associated protein tau in liquid-liquid phase separated condensates revealed by NMR spectroscopy. (A) Liquid-droplets of tau visualized by fluorescence microscopy. (B) Paramagnetic broadening in 2D 1H-13C HSQC spectra of the microtubule-binding domain of tau at 5°C (left; dispersed phase) and 37°C (right; phase separated conditions) as seen for its four threonine residues. The microtubule-binding domain of tau was tagged with MTSL at its two native cysteines. Paramagnetic and diamagnetic states are represented by gold and black color, respectively. Reprinted from ref. [2] under a Creative Commons license (CC BY 4.0, http://creativecommons.org/licenses/by/4.0/).
Figure 2: Using NMR to investigate the properties of the A182–S197 region of the nucleocapsid (NCP) protein within SARS-CoV-2, which has a high proportion of serine and arginine resides (known to bind both RNA and proteins). (A) Chemical shift analysis (blue) agrees with molecular dynamics simulations (red) that the residues in this region are very flexible, with a small propensity for α-helical structure next to R189. (B) Re-running the simulations in the presence of polyuridylic acid (a simplified RNA) showed a large number of intermolecular contacts between the arginine residues and the RNA phosphate groups, with a maximum for R189. This agrees with the observation that R189 is the only residue in the region A182–S197 that is not mutated in most of the currently known strains of SARS-CoV-2 (gray bars). Reprinted from ref. [4] under a Creative Commons license (CC BY 4.0, http://creativecommons.org/licenses/by/4.0/).

The 1.2 GHz Bruker instrument at Göttingen

The work of Prof. Dr. Zweckstetter at Göttingen has involved a number of high-field NMR instruments from Bruker. The latest addition to this portfolio of equipment is a 1.2 GHz instrument, which was installed in the middle of 2020. It is the NMR spectrometer with the highest magnetic field available world-wide to study the structure and dynamics of biomolecules.
The magnetic coil in the instrument is unique to Bruker and uses high-temperature superconductors for the inner coil and regular, low-temperature superconductors for the outer coil. It is only in this way that the uniform magnetic field of 28.2 Tesla can be generated.
Four teams at Göttingen have access to the 1.2 GHz instrument, with its key function being to characterize proteins that are difficult to study with other methods, such as those in membranes and those with a tendency to aggregate, like those implicated in neurodegeneration.

Highest resolution, exciting possibilities

Until recently, most of the team’s work used Bruker NMR spectrometers from 600 MHz to 950 MHz, which have been central to carrying out the highly sensitive investigations into proteins. But Prof. Zweckstetter now has access to the 1.2 GHz instrument at Göttingen, which “will enhance the resolution of our three- and higher-dimensional NMR experiments by at least a factor of two compared to our existing 950 MHz instrument, and so allow us to study the structural dynamics of biomolecular markers on huge range of time and length scales”, he says.

With this boost to NMR science at Göttingen, Prof. Zweckstetter is enthusiastic about future possibilities: “One aspect we are keen to pursue is to integrate NMR with other structural biology techniques, and so obtain a more complete view of the inner workings of the SARS-CoV-2 virus. For example, our studies have highlighted the key role of N protein in the virus, and further characterization using high-resolution NMR, molecular dynamics simulations and phase separation experiments should help us pinpoint how potential drugs interact with N protein, and whether RNA replication could be influenced”.

The capabilities of high-field NMR stand in contrast, he says, to techniques such as cryoelectron microscopy, which requires frozen samples, and so can only provide snapshots of the molecular action: “With NMR, you can study how the biomolecule moves around in solution, and the different shapes it takes on to perform different activities”. Not only that, he says, but you can visualize molecules in real-time, and so gain crucial insights into how they perform their function and are modified by enzymes.

Collaboration versus competition

The Covid-19 NMR project has given Prof. Zweckstetter pause for thought about the wider role of research and society today. “Collaboration is of course valued in science, but the inherent competition between research groups limits the scope of that collaboration”, he says.

But the Covid-19 consortium has completely changed the importance that he attributes to collaboration: “Whereas we might ordinarily work just with one or two groups, we now have the opportunity to regularly discuss our progress with dozens of research teams globally. By pooling our expertise, equipment and reagents, we can make progress much more quickly, and potentially achieve so much more than we could on our own”.
The end result of this work, he hopes, will be better treatment for those with Covid-19: “While vaccination is very important, this virus is constantly adapting and evolving, bringing a risk of re-infection. So to counter the threat it poses, we need to develop drugs to treat Covid-19 – and this is where the consortium comes in, by providing the data that enables us to understand the inner workings of the virus.”

In fact, he believes that it is important to re-think how research is done: “Academic competition has the potential advantage that the best ideas might win. On the other hand, tight competition puts enormous pressure on researchers, especially those in the early stages of their careers, and in addition it doesn’t necessarily generate the best results in the shortest time-frame. So, it’s my view that setting up large scientific collaborations to tackle major challenges could be a complementary and maybe even more powerful way of making rapid progress.”

The Covid-19 NMR work certainly seems to have inspired Prof. Zweckstetter to deepen his links with other members of the consortium, and more widely too. And he would encourage others to think along similar lines, concluding: “Perhaps science should be more collaborative, and less competitive.”

For more information on the Covid19-NMR consortium, please visit https://covid19-nmr.de/ or watch the interviews with the consortium members https://www.bruker.com/en/products-and-solutions/mr/make-mr-more-relevant/covid19-nmr-consortium.html

For more information about the “Structure determination of proteins using NMR” and “Translational Structural Biology” research groups of Prof. Zweckstetter, please visit https://www.mpibpc.mpg.de/zweckstetter and https://www.dzne.de/zweckstetter.
 

Prof. Dr. Markus Zweckstetter (left) and Prof. Dr. Christian Griesinger in front of the 1.2 GHz Bruker instrument, upon its arrival at Göttingen in summer 2020.

References:

  1. Jaremko Ł, Jaremko M, Giller K, Becker S and Zweckstetter M, Structure of the mitochondrial translocator protein in complex with a diagnostic ligand, Science, 2014, 343: 1363–1366, https://doi.org/10.1126/science.1248725.
  2. Ambadipudi S, Biernat J, Riedel D, Mandelkow E, Zweckstetter M, Liquid-liquid phase separation of the microtubule-binding repeats of the Alzheimer-related protein Tau, Nat Commun, 2017, 17;8(1):275. doi: 10.1038/s41467-017-00480-0.
  3. For more about the Covid-19 consortium, visit https://covid19-nmr.de/
  4. Savastano A, Ibanez de Opakua A, Rankovic M and Zweckstetter M, Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates, Nature Communications, 2020, 11: 6041, https://doi.org/10.1038/s41467-020-19843-1
     

About the Max Planck Institute for Biophysical Chemistry

The Max Planck Institute for Biophysical Chemistry currently consists of 11 departments, 8 Emeritus groups and 21 research groups with their own research focus. These are supported through 15 central scientific and general service facilities. With more than 700 people – among them about 470 scientists – our institute is one of the largest in the Max Planck Society and unique in its multidisciplinary approach.

About the German Center for Neurodegenerative Diseases (DZNE)

The DZNE was founded in 2009 as a member of the Helmholtz Association and the first member of the German Centers for Health Research (German Coeliac Society, DZG). Today, it consists of ten sites – Berlin, Bonn, Dresden, Göttingen, Magdeburg, Munich, Rostock/Greifswald, Tübingen, Ulm and Witten – and consequently pools expertise distributed nationwide within a single research institute. More than 1,100 experts are working to understand what causes diseases of the brain and nervous system and to find new approaches for effective prevention, therapy, and patient care. Worldwide, the DZNE is one of the largest research institutes dealing with this topic.

About Bruker Corporation

Bruker is enabling scientists to make breakthrough discoveries and develop new applications that improve the quality of human life. Bruker’s high-performance scientific instruments and high-value analytical and diagnostic solutions enable scientists to explore life and materials at molecular, cellular and microscopic levels. In close cooperation with our customers, Bruker is enabling innovation, improved productivity and customer success in life science molecular research, in applied and pharma applications, in microscopy and nanoanalysis, and in industrial applications, as well as in cell biology, preclinical imaging, clinical phenomics and proteomics research and clinical microbiology.