Conflicting Interfacial Electrostatic Interactions as a Design Principle to Modulate Long-Range Interdomain...

Area of Science:

• Biochemistry
• Structural Biology
• Protein Dynamics

Background:

• Interdomain communication in multidomain proteins is crucial for function and allostery.
• Electrostatic interactions at domain interfaces can mediate interdomain connectivity.
• The FF3-FF4 domain of p190A RhoGAP protein serves as a model system.

Purpose of the Study:

• To investigate how electrostatic interactions at the FF3-FF4 interface influence protein stability, folding, and dynamics.
• To explore the role of a conserved charge network in shaping the protein's native ensemble.
• To understand design principles for engineering proteins with enhanced interdomain communication.

Main Methods:

• Site-directed mutagenesis (charge-reversal mutation).
• Protein stability and folding kinetics measurements (e.g., circular dichroism, fluorescence).
• Computational modeling to analyze electrostatic interactions and conformational landscapes.

Main Results:

• Wild-type FF34 exhibits slow folding, low cooperativity, and distinct stabilities due to a frustrated electrostatic network (charge troika) at the FF3-FF4 interface.
• This network promotes multiple, decoupled structural states on a rugged energy landscape.
• A charge-reversal mutation disrupts the frustrated network, significantly enhancing stability and cooperativity, and accelerating folding by over an order of magnitude.
• The mutation globally dampens fluctuations.

Conclusions:

• Conserved, nonoptimal interfacial electrostatic interactions shape the native ensemble of bilobed proteins.
• Modulating these interactions can control protein stability, cooperativity, and folding dynamics.
• This work provides a foundation for designing molecular systems with tunable long-range connectivity and cooperativity.