Shelterin is a DNA-binding complex that facilitates telomeric DNA replication and maintenance of telomeric stability 1. Mutations in shelterin components can lead to various pathologies, including cancer predisposition and premature aging. While individual structured domains of shelter have been characterized, there are no high-resolution structural models for full-length shelterin proteins 2.
A recent study employs techniques like mass photometry, negative stain EM, structural modeling and native mass spectrometry to characterize shelterin subcomplexes and their structural variability 2. The findings reveal a high level of conformational heterogeneity in shelterin's structure, which has implications for its diverse functions.
To gain further insight into this research, we spoke to the first author of this study, John Zinder, PhD – formerly a postdoc in Titia De Lange’s and Thomas Waltz’s laboratories at Rockefeller University and currently a senior scientist at Odyssey Therapeutics.
Keep reading and register for a free webinar where John will discuss how the shelterin complex was reconstituted and how he used mass photometry to study the complex’s extensive conformational heterogeneity.
The interview responses have been edited for length and clarity.
Outline
Introducing John Zinder – the study’s first author
John Zinder is currently a Senior Scientist in the Structural Biology group at Odyssey Therapeutics and works in the development of novel therapeutics. He has an undergraduate degree in chemistry (Cornell University) and a master’s degree in biochemistry (University of Colorado). He was awarded a PhD in chemical biology from his research on the S. cerevisiae nuclear RNA exosome in Chris Lima’s lab at the Sloan Kettering Institute. He subsequently became a Damon Runyon Postdoctoral Fellow in the de Lange and Walz labs at the Rockefeller University.
Aim: Understand the architecture & stoichiometry of the shelterin complex
Human shelterin is a six-subunit complex, composed of the TRF1, TRF2, Rap1, TIN2, TPP1, and POT1 proteins. Even though the individual subunits have been structurally and functionally characterized, there are currently no high-resolution structural models for full-length shelterin proteins – possibly due to shelterin’s flexible structure, that serves in implementing diverse functions in telomeres. Therefore, the aim of this study was to reconstitute the full shelterin complex, by assembling the purified subunits in vitro, and to study the complex’s architecture and stoichiometry 2.
John explains: “Since the discovery of the first telomeric protein, TRF1and subsequent description of the other five components, every protein in the shelterin complex has been intensely structurally and biochemically scrutinized 1, 3. When I came to the project, we had high-resolution structures of every domain within the complex and of key interactions for the complex’s assembly. The question was what we can learn about telomere protection by putting full-length proteins together to form complexes that would exist inside our cells.”
Conformational variability & stoichiometry of the shelterin complex
The TRF1 complex
To assess conformational heterogeneity, John and his colleagues, first purified a complex containing POT1, TPP1, TIN2, and TRF1, referred to as the TRF1 complex. Using mass photometry, they observed four distinct peaks corresponding to different mass values. These peaks represented different compositions within the complex including two, three, five and eight individual subunits 2.
Then, they aimed to study the stability of the dimeric complex by adding telomeric DNA (TeloDNA1). In the presence of DNA, the purified TRF1 complex appeared as a single species with a mass close to the predicted molecular mass of the eight-component dimer-of-tetramer complex bound to DNA 2.
The full shelterin complex
Subsequently, John and his colleagues reconstituted the full shelterin complex by mixing purified TRF2/Rap1 with the TRF1 complex, in the presence and absence of telomeric DNA (TeloDNA2). Mass photometry analysis of the full complex revealed three prominent peaks at 300, 495, and 780 kDa, indicative of different possible stoichiometric arrangements. The addition of TeloDNA2 resulted in shifts in the peaks, further supporting the assigned stoichiometries 2.
Based on the mass photometry analysis, the researchers concluded that shelterin can assemble into a fully dodecameric (or dimer-of-hexamers) complex. The presence of peaks corresponding to the TRF1 octameric complex and the full shelterin dodecameric complex supported this conclusion 2.
In addition, the mass photometry data show the presence of conformational heterogeneity in the shelterin complex 2. When asked about the biological advantage of this variability, John comments: “We tend to think of proteins as adopting a single confirmation or a small subset of discrete conformations, in part because structural biology has only recently acquired the tools to observe this type of flexibility. However, a vast amount of the proteome contains all these flexible, intrinsically disordered regions, and it’s likely that most complexes in the cell exhibit some sort of continuous flexibility that we're only just beginning to scratch the surface of. Particularly with shelterin, I view its dynamic nature as being an advantage. Telomeres are dynamic entities, and shelterin may need to adopt a vast array of confirmations to perform its distinct telomere-specific functions.”
Orthogonal use of mass photometry, native MS and negative-stain EM
In this study, mass photometry provided valuable information about the compositional heterogeneity, stoichiometry, and stability of the shelterin complex. It complemented other techniques such as native mass spectrometry and negative-stain EM, contributing to a comprehensive understanding of shelterin's structural characteristics and assembly 2.
Each of these techniques gave John and his colleagues' valuable information. Mass photometry gave insights into the negative stain electron microscopy data. John explains:
“In the beginning, we were using a low-resolution electron microscopy technique called negative staining EM. We observed a high level of heterogeneity for the TRF1 complex but had no way of determining whether it arose from different conformations within the sample or different stoichiometries. With mass photometry, when DNA was added, the TRF1 complex collapsed to the single monodisperse species. Then we could say with confidence that there was only one discrete stoichiometry in solution, despite seeing many different shapes in negative stain EM. This gave us confidence that conformational variability rather than compositional heterogeneity gave rise to the heterogeneity we were seeing.”
Furthermore, both mass photometry and native MS confirmed the dimeric state of shelterin. John comments: “We really wanted to prove that the dimeric stoichiometry of shelterin was possible and were able to collaborate with Dominic Olinares in Brian Chait’s group and do native MS. With both methods, we were able to assign an accurate dimer peak, but also show all the other species”.
The benefits of using mass photometry in structural biology
Compared to all the techniques that John used in this study, he was surprised about how fast and easy a mass photometry experiment can be. He comments:
“Compared to the other methods for looking at compositional heterogeneity, mass photometry is incredibly quick and requires very little sample. I could just take an hour out of my day to do a bunch of experiments. It didn't really require extensive expertise and gave me an incredible amount of flexibility.”
He also claimed that the fact that mass photometry measurements happen in solution, is very beneficial for his structural biology questions. John explains:
“I feel that mass photometry is really reporting on my actual sample as I have it in my Eppendorf tube. That's a huge benefit. In particular, you can parse out compositional heterogeneity independent of conformational variability, which is a difficult thing to get with other techniques. With cryo-EM, for example, you see heterogeneity sometimes, but you don't know if that's because of the conformational variability, compositional variability, or sample denaturation during vitrification.”
Finally, John was surprised about how intuitive the mass photometry analysis software (DiscoverMP) was. He explains:
“The mass photometry analysis software is super easy to use. It can give you an idea of what is happening right off the bat so you could course-correct. For example, I could instantly see if I was over concentration or under concentration. This is not the same with other techniques, such as cryo-EM. With cryo-EM, I had to set up a bunch of grids and then, depending on microscope availability, find out weeks later that I'm tenfold too concentrated.”
Looking ahead – next steps for shelterin research
The combination of mass photometry, negative-stain electron microscopy, structural modeling and mass spectrometry have significantly advanced our understanding of shelterin's structure and dynamics. These techniques have revealed the complex's flexibility, heterogeneity, and stoichiometry, providing crucial insights into how shelterin interacts with telomeric DNA and other factors involved in telomere maintenance. By unraveling the intricate details of shelterin, these findings pave the way for further investigations into telomere biology and potential therapeutic strategies targeting telomere-related diseases.
Further resources
Read our technical blog explaining the principle behind mass photometry and why mass photometry is useful.
Here you can learn how mass photometry directly measures the relative concentrations of all protein populations in a sample, in a single-molecule fashion.
In this webinar, Dr. Margaret Stratton (UMass Amherst) talks about her work on the role of CaMKII in different tissues. Mass photometry has been used to quantitatively determine the stoichiometry of different variants of this crucial oligomeric enzyme.
References
(1) De Lange, T. Shelterin: The Protein Complex That Shapes and Safeguards Human Telomeres. Genes Dev. 2005, 19 (18), 2100–2110. https://doi.org/10.1101/gad.1346005.
(2) Zinder, J. C.; Olinares, P. D. B.; Svetlov, V.; Bush, M. W.; Nudler, E.; Chait, B. T.; Walz, T.; de Lange, T. Shelterin Is a Dimeric Complex with Extensive Structural Heterogeneity. Proceedings of the National Academy of Sciences 2022, 119 (31), e2201662119. https://doi.org/10.1073/pnas.2201662119.
(3) Chong, L.; Van Steensel, B.; Broccoli, D.; Erdjument-Bromage, H.; Hanish, J.; Tempst, P.; De Lange, T. A Human Telomeric Protein. Science 1995, 270 (5242), 1663–1667. https://doi.org/10.1126/science.270.5242.1663.