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Decoding Coupled Folding and Binding via Single-Molecule characterization of Ancient IDP protein-protein Interactions

Periodic Reporting for period 1 - evoDISFOLD (Decoding Coupled Folding and Binding via Single-Molecule characterization of Ancient IDP protein-protein Interactions)

Reporting period: 2019-09-01 to 2021-08-31

Project Context:
Intrinsically Disordered Proteins (IDPs) lack a defined 3-dimensional structure under physiological conditions, and can perform their functions as disordered ensembles. They are now acknowledged as extremely relevant players of cellular processes because of their abundance in proteomes, and also because they are associated with crucial functions of signaling and regulation. The historical neglect of IDPs is in part explained by the limitations of classical biophysical techniques for resolving IDPs conformational heterogeneity and dynamics. Nonetheless, in the last decade single molecule techniques like fluorescence spectroscopy using Förster Resonance Energy Transfer (smFRET) have greatly advanced the understanding and modelling of IDPs complexity.
Some IDPs develop their activity as fully disordered ensembles, but many also undergo different degrees of disorder-to-order transition when they bind other molecules to perform their function—this process is known as coupled folding and binding. A widely studied example is that of the nuclear co-activator binding domain of transcriptional co-activators CREB-binding protein and p300 (NCBD), upon interaction with its multiple binding partners.

Overall Objectives:
Our group pioneered the study of IDPs using smFRET—in particular, it has extensively studied the coupled folding and binding of NCBD with several of its partners, some also intrinsically disordered like the CREBBP interacting domain of Nuclear Receptor Coactivators 3 (ACTR) and 1 (SCR-1), or the transactivation domain of p53 (p53TAD).
The overall objective of the project was to expand the possibilities to study IDPs with single-molecule techniques by developing methods and protocols allowing to use Optical Tweezers force spectroscopy to analyze the coupled folding and binding of NCBD with its interaction partners. With this achievement, we sought to obtain new orthogonal information and to progress towards the integration of high resolution force and smFRET data—with the ultimate goal of disentangling general principles of coupled folding and binding.
Work Performed:
To attain our objectives, we first engineered protein constructs allowing to analyze, using Optical Tweezers, the coupled folding and binding of NCBD with its interaction partners—so we designed and produced constructs of NCBD fused with its corresponding binding partner through a polypeptide linker. Next, we optimized protocols to perform measurements on our fusions using Optical Tweezers by attaching the protein constructs to microspheres via DNA handles. Finally, we also developed specific methods to analyze and interpret the wealth of data obtained from different types of Optical Tweezers measurements.

Main Results:
In constant velocity measurements, the coupled folding and binding of NCBD with ACTR or SRC-1 is observed as a clear transition in Force-extension traces. In contrast with typical folding/unfolding transitions of structured proteins—more frequently detected as discrete jumps—for those protein fusions, coupled folding and binding transitions are observed as a highly dynamic exchange between a high-force state (associated with the fully bound and folded complex) and a low-force state (associated with the unbound and unfolded interactors) in the low picoNewtons. Conversely, for the fusion of NCBD with p53TAD no clear transition is observed, but coupled folding and binding could still be perceived as a subtle kink in Force-extension traces also in the low picoNewtons. The difference observed, depending on the specific interactor, is consistent with the known lower affinity of p53TAD to bind NCBD.
We could also record the kinetics of coupled folding and binding of NCBD with either ACTR or SRC-1 in constant distance measurements for long periods of time—for several minutes up to an hour. The resulting traces revealed the coupled folding and binding of NCBD with either ACTR or SRC 1 is characterized by very fast transitions between apparently only two states: the fully bound and folded (high force) and the completely unbound and unfolded (low force). However, a thorough analysis of kinetic data shows the disorder to order transitions of these protein complexes cannot be explained by a simple two state model—but requires to consider at least two kinetic regimes, involving the population of additional states. This finding is in agreement with previous results from our group using smFRET . More relevantly, our experiments using Optical Tweezers revealed a completely novel feature of the coupled folding and binding of NCBD with either ACTR or SRC-1 where the interaction partners dwell in an unbound and unfolded, apparently non productive or “locked”, state for tens of milliseconds to several seconds.
Given the role of Proline isomerization in the kinetic behavior previously evidenced with smFRET, we designed a collection of Proline mutants to survey the effect of their isomerization on the kinetic features observed under force . As in smFRET experiments, a specific Proline seems to mainly govern the switching between kinetic regimes—but using Optical Tweezers we find that its mutation does not fully prevent the flux between the two regimes, suggesting a minor involvement of other Prolines. Interestingly, the absence of Prolines does not preclude the population of the “locked” state; but some Prolines seem to influence the frequency and lifetime of its population.
Since Proline isomerization alone could not explain the population of this new “locked” state, we evaluated (focusing on the NCBD-ACTR model) whether it could result from specific properties of the single interacting partners—so we studied NCBD and ACTR alone using Optical Tweezers. Interestingly, while ACTR does not present any detectable transition in constant velocity measurements, NCBD shows a subtle but clearly recognizable transition in Force extension traces. We could also record kinetics of the intrinsic transitions of NCBD in constant distance measurements, and we observe that NCBD populates a lower force state with a lifetime in a range of few seconds. We hypothesize these transitions may correspond to the full unfolding of the Molten Globule conformation NCBD is considered to populate in isolation, and that disruption of this Molten Globule may explain the population of the “locked” state by the complex fusions.
Progress beyond State-of-the-Art and Potential Impact:
To our best knowledge, our results represent the first characterization of coupled folding and binding upon interaction between IDPs using Optical Tweezers. Our analysis of the coupled folding and binding using the NCBD model is consistent with previous smFRET findings; but, more importantly, our innovative application allowed to unveil and characterize a previously unresolved feature of this system. Our results suggest that maintenance of a partially structured conformation by NCBD in its unbound state is essential for a productive coupled folding and binding with its interaction partners. It will be extremely interesting to use our approach to study whether this feature could also explain the behavior of other systems.
Globally, we have demonstrated the feasibility of using Optical Tweezers to study physiologically relevant conformational transitions of IDPs, at very low forces. The methods and protocols we have developed are important contributions to the experimental toolbox for the study of IDPs.
Dumbbell configuration for the study of biomolecules using dual-trap Optical Tweezers.