Walking the Tightrope: Balancing Opposing Cooperativities as an Operating Principle in Dynein Assembly

Area of Science:

• Molecular Biology
• Biophysics
• Systems Biology

Background:

• The central region of cytoplasmic dynein, crucial for cargo transport, involves a complex seven-state binding network.
• Traditional methods like isothermal titration calorimetry (ITC) struggle to quantitatively analyze such intricate molecular interactions.
• Understanding this network is key to deciphering dynein's role in cellular processes.

Purpose of the Study:

• To develop a novel quantitative approach for characterizing complex, multi-state protein binding interactions.
• To precisely determine the thermodynamic landscape of the cytoplasmic dynein intermediate chain and light chain assembly.
• To uncover previously unrecognized binding states and regulatory mechanisms in dynein function.

Main Methods:

• Combined extensive experimental sampling (39 ITC isotherms) with hierarchical Bayesian inference.
• Developed a global Bayesian framework to integrate multiple datasets and account for concentration uncertainty.
• Fitted data to a mechanistic seven-state model, estimating 190 parameters with high precision.

Main Results:

• Achieved highly precise thermodynamic parameter estimates (95% confidence intervals as narrow as 0.05 kcal/mol).
• Enabled back-propagation to nanomolar precision in effective concentrations, even from high experimental concentrations.
• Revealed a form of "multi-cooperativity" governing dynein assembly, including previously inaccessible binding states.

Conclusions:

• The developed Bayesian approach can resolve complex molecular systems beyond the capabilities of traditional biophysical techniques.
• The findings reveal new binding states in dynein assembly potentially critical for cargo attachment and release.
• Demonstrated that negative cooperativity is a key regulatory strategy for tuning molecular responsiveness and dynamic control.