Authors: Sebastian Fielder1, Monika Pizioska1, Christian Mayners2, Felix Hausch2, Sean Devenish1, Heike Fiegler1, Tuomas Knowles3,4
1Fluidic Analytics, Cambridge, United Kingdom
2Department of Chemistry, Clemens-Schöpf-Institute for Organic Chemistry and Biochemistry Technische Universität Darmstadt, Germany
3Centre for Misfolding Diseases, Yusuf Hamied Department of Chemistry, University of Cambridge, United Kingdom
4Cavendish Laboratory, Department of Physics, University of Cambridge, United Kingdom
Decades of research have led to a remarkable understanding of protein structure and function. However, proteins rarely act on their own and a quantitative understanding of their interaction mechanisms is key to the successful development of new drugs that enable the modulation of the cellular network of protein–protein interactions. In the study presented here we have quantified the effect of rapamycin, a small molecule immunosuppression drug, on the protein complex formation of FKBP12 and mTOR using an in-solution assay that enables the characterization of otherwise challenging multi-protein complexes under close to native conditions.
The ability to reduce the activity of the immune system (immunosuppression) in a controlled fashion represents one of the most important achievements in modern medicine.
Without immunosuppression, organ transplants, bone-marrow transplants, or the treatment of auto-immune diseases such as rheumatoid arthritis or Crohn’s disease would not exist. Many immunosuppressant drugs modulate the delicate network of protein–protein interactions within the body. Rapamycin, a small molecule drug, for example, acts as an immunosuppressor by targeting the FK506-binding protein (FKBP12) to create a new interaction surface.1 This modification allows FKBP12 to form a complex with mammalian TOR (mTOR) which reduces the sensitivity of T- and B-cells to interleukin-2 and attenuates the immune reaction.
While we can name the interaction partners of many drugs and drug candidates, surprisingly, we often only have little knowledge about the underlying mechanisms via which these drugs and drug candidates interact with their intended targets. A quantitative understanding of these mechanisms, however, is key for successful drug development.
Conventional technologies including SPR (Surface Plasmon Resonance) and BLI (Biolayer Interferometry), though, having made significant contributions to drug development, often struggle when characterizing more challenging interactions such as formation of multi-protein complexes. As both technologies rely on attaching one of the binding partners to a surface, this very feature can become an obstacle as the surface-immobilization is likely to interfere with the measurements. This in turn might make it more difficult to distinguish whether a drug or drug candidate binds to a monomeric protein, a misfolded protein, or a multi-protein complex. Another problem with most surface-based technologies is that these methods rely on measuring binding kinetics to determine binding affinity.
For heterogenous protein targets such as complexes composed of several proteins, the binding kinetics can become increasingly complicated which makes it challenging to extract any meaningful quantitative information about the binding reaction and its stoichiometry, specifically without any additional information on complex composition or size.
In the study presented here, we have characterized the rapamycin-induced protein complex formation of FKBP12 and mTOR using an in-solution assay that ;overcomes the limitations presented by surface-based technologies. With this approach, we were able to determine the affinity (KD) as well as the stoichiometry of the protein complex. The assay can be easily adopted to other protein targets for the study of otherwise difficult to characterize multi-protein complexes.
In the presence of rapamycin and with increasing concentrations of FRB, we observed an increase in the hydrodynamic radius, Rh, of FKBP12-FITC, indicating ternary complex formation (Figure 1). At FRB concentrations > 100 nM, the Rh remained largely constant at around 2.6 nm, which indicates binding saturation. In contrast, a control sample composed of 20 nM FKBP12-FITC and 1 μM FRB that lacked rapamycin displayed an Rh of 1.92 nm, similar to that of unbound FKBP12-FITC. The KD was determined to be 13 nM (95% CI: 4 – 33), in line with literature values.4
Figure 1: Rapamycin-mediated binding of FKBP12-FITC to FRB. By use of the Fluidity One-W, the stoichiometry of a protein complex can be inferred based on its size. To do so, the experimental Rh of the FKBP12/FRB complex is compared with a predicted Rh. The Rh prediction is provided by the hydrodynamic radius converter on the Fluidic Analytics website and utilizes the individual molecular weights of FKBP12 (14.6 kDa) and FRB (14.7 kDa). The predicted Rh for a 1:1 binding stoichiometry of FKBP12 and FRB (i.e., a 29.3 kDa complex) agrees very well with the measured Rh, as shown in Figure 2.
Figure 2: Confirmation of 1:1 stoichiometry for the binding of FKBP12 to FRB in the presence of rapamycin. Prediction of Rh for a 1:1 complex is based on nominal molecular weights of 14.6 kDa and 14.7 kDa of FKBP12 and FRB, respectively, resulting in a complex of 29.3 kDa. This nominal molecular weight of the complex was entered into the hydrodynamic radius converter on the Fluidic Analytics website to retrieve the predicted Rh.5 The contribution of rapamycin (0.9 kDa) to the Rh of the protein complex is within the error of the method.
We describe an in-solution assay on the Fluidity One-W platform to characterize the interaction of the small-molecule drug rapamycin with its interaction partners FKBP12 and mTOR. This approach allowed us to determine the affinity (KD) as well as the stoichiometry of the formed protein complex. As the assay is based on in-solution, equilibrium binding measurements, surface effects are eliminated, and complex binding kinetics, commonly observed for multicomponent systems, do not need to be considered.
Moreover, as the assay requires only one of the proteins involved in the complex to be fluorescently labeled, it can easily be adapted to other multi-protein systems to characterize the mechanisms of interactions between drug or drug candidates and their intended targets for successful drug development.
- Li, J.; Kim, S. G.; Blenis, J. Rapamycin: One Drug, Many Effects. Cell Metabolism, 2014, 19, 373. DOI: 10.1016/j.cmet.2014.01.001
- Hähle, A.; Geiger, T. M.; Merz, S.; Meyners, C.; Tianqi, M.; Kolos, J.; Hausch, F. FKBP51 and FKBP12.6—Novel and tight interactors of Glomulin. PLoS ONE, 2019, 14 e0221926. DOI: 10.1371/journal.pone.0221926
- März, A. M.; Fabian, A. K.; Kozany, C.; Bracher, A.; Hausch, F. Large FK506-binding proteins shape the pharmacology of rapamycin. Mol Cell Biol., 2013. 33 (7), 1357-1367. DOI: 10.1128/mcb.00678-12
- Kostrz, D.; Wayment-Steele, H. K.; Wang, J. L.; Follenfant, M.; Pande, V. S.; Strick, T. R.; Gosse, C. A modular DNA scaffold to study protein–protein interactions at single-molecule resolution. Nature Nanotechnology, 2019, 14, 988. DOI: 10.1038/s41565-019-0542-7
- Tyn, M.T.; Gusek, T. W. Prediction of Diffusion Coefficients of Proteins. Biotechnology and Bioengineering, 1990, 35, 327. DOI: 10.1002/bit.260350402
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