Direct Observation of Small Molecule Activator Binding to Single PR65 Protein

Chemistry Medicinal Chemistry Life Sciences Medicine Biochemistry Biophysics
Protein Phosphatase 2A Small Molecule Activator Nanoaperture Optical Tweezers Binding Kinetics Molecular Dynamics Simulation

Single-Molecule Optical Tweezer Technique Reveals the Binding Mechanism of a Small Molecule Activator and PR65 Protein

Academic Background

Protein phosphatase 2A (PP2A) is a crucial enzyme in regulating cellular signaling. Its dysfunction is closely associated with various cancers and chronic diseases, such as Alzheimer’s disease and chronic obstructive pulmonary disease. Therefore, reactivating PP2A has been recognized as a key strategy for treating these conditions. In recent years, small molecule activators (SMAPs) have been developed to directly bind to the scaffolding subunit PR65 of PP2A, thereby restoring its functionality. However, the binding mechanism between PR65 and SMAPs, as well as their impact on protein conformation, remains unclear. To address this issue, researchers employed single-molecule optical tweezers (NOTs) combined with molecular dynamics (MD) simulations to study the binding kinetics of the small molecule activator ATUX-8385 with PR65 and its effects on protein conformation.

Source of the Paper

This paper was jointly authored by a research team led by Annie Yang-Schulz, Maria Zacharopoulou, Semazeynep Yilmaz, and others from institutions such as the University of Victoria, the University of Cambridge, and Stony Brook University. It was published in npj Biosensing in 2025. The research was supported by multiple grants, including HFSP (RGP0027/2020) and BBSRC (BB/T002697/1).

Research Process and Results

1. Research Process

a) Single-Molecule Optical Tweezer Experiments

Researchers utilized dual-nanohole optical tweezers (DNH-NOTs) to directly observe the binding process between ATUX-8385 and PR65 under label-free conditions. The experimental procedure included: 1. Sample Preparation: PR65 protein was mixed in a 5% DMSO solution with 20 μM of ATUX-8385. 2. Optical Tweezers Capture: A single PR65 protein molecule was captured using a focused laser beam on the dual nanohole. 3. Signal Recording: Changes in light scattering signals of the protein before and after binding were recorded using an avalanche photodiode (APD). 4. Data Analysis: The signals were analyzed using a hidden Markov model (HMM) to distinguish between bound and unbound states, and association and dissociation rate constants were calculated.

b) Molecular Dynamics Simulations

To validate the experimental results, researchers conducted molecular dynamics simulations: 1. Docking Simulations: Using Rosetta and AutoDock Vina software, ATUX-8385 was docked onto PR65 to identify potential binding sites. 2. Molecular Dynamics Simulations: Apo-PR65 and ATUX-8385-bound PR65 were simulated for a total of 4.224 microseconds, analyzing protein conformational changes. 3. Verification of Binding Sites: The simulations showed that ATUX-8385 stably bound to the 4th and 5th helical repeat regions of PR65 and favored an extended conformation of PR65.

c) Biophysical Characterization

To further verify the binding characteristics, researchers utilized the following techniques: 1. Nano Differential Scanning Fluorimetry (nanoDSF): Measured changes in the melting temperature ™ of PR65 in the presence of ATUX-8385, indicating increased protein stability upon binding. 2. Fluorescence Polarization Experiments: Determined the dissociation constant (Kd) of ATUX-8385 and PR65 interaction using fluorescence polarization, yielding a value of 9.4 ± 1.4 μM, consistent with single-molecule experimental results. 3. Nuclear Magnetic Resonance (NMR): Used 19F NMR to detect the binding of ATUX-8385 to PR65, further confirming the binding event.

2. Key Results

a) Single-Molecule Experimental Results

  • Binding Kinetics: Using the NOTs technique, the dissociation constant (Kd) of ATUX-8385 and PR65 was determined to be 13.6 ± 2.5 μM, consistent with fluorescence polarization experiments.
  • Conformational Changes: Upon ATUX-8385 binding, PR65 exhibited a significant increase in light scattering signals, indicating a conformational shift from a compact state to an extended state.

b) Molecular Dynamics Simulation Results

  • Binding Site: ATUX-8385 stably bound at the 4th and 5th helical repeats of PR65, providing reliability to the experimental observations.
  • Conformational Stability: Simulations showed that ATUX-8385 binding favored PR65’s extended conformation, aligning with the experimental increase in light scattering signal.

c) Biophysical Characterization Results

  • nanoDSF: PR65’s melting temperature increased from 52.7 ± 0.1 °C to 53.5 ± 0.1 °C in the presence of ATUX-8385, indicating enhanced protein stability.
  • Fluorescence Polarization: The determined Kd value of 9.4 ± 1.4 μM highly agreed with single-molecule experimental data.
  • NMR: Changes in 19F NMR signal intensity confirmed ATUX-8385’s binding to PR65.

3. Conclusions and Significance

This study combined single-molecule optical tweezers with molecular dynamics simulations to reveal, for the first time, the binding mechanism between ATUX-8385 and PR65 at the single-molecule level, as well as its effects on protein conformation. The results suggest that ATUX-8385 promotes PP2A reactivation by stabilizing the extended conformation of PR65. These findings provide new molecular insights into PP2A-targeted therapies and demonstrate the significant potential of single-molecule optical tweezers in drug discovery.

4. Research Highlights

  • Single-Molecule Level Study: Observed the binding kinetics between ATUX-8385 and PR65 for the first time at the single-molecule level.
  • Multi-Technique Validation: Combined NOTs, MD simulations, nanoDSF, fluorescence polarization, and NMR, ensuring comprehensive validation of the findings.
  • Applications in Drug Discovery: Demonstrated the value of single-molecule optical tweezers for studying drug binding kinetics and screening.

5. Additional Valuable Information

  • Temperature Modulation: The NOTs technique allows for local temperature modulation by adjusting laser power, enabling future studies on temperature effects on binding kinetics.
  • Open Data: Research data and code are publicly available on GitHub (https://github.com/nanoplasmonics/smaps) for verification and further exploration by other researchers.

Summary

This study utilized innovative single-molecule optical tweezer techniques to reveal the binding mechanism of the small molecule activator ATUX-8385 with the PR65 protein and its effects on protein conformation. This advancement not only deepens the understanding of PP2A reactivation mechanisms but also provides new tools and methodologies for drug discovery.