Engineering rotor ring stoichiometries in the ATP synthase

Area of Science:

• Biochemistry
• Molecular Biology
• Bioenergetics

Background:

• ATP synthase membrane rotors are composed of c-subunit rings with varying stoichiometry across species.
• Understanding the factors influencing c-subunit ring size is crucial for deciphering ATP synthase function and diversity.

Purpose of the Study:

• To investigate the role of conserved glycine residues in c-subunit contacts and their impact on rotor stoichiometry.
• To explore the functional consequences of altered c-ring stoichiometry on ATP synthesis efficiency.

Main Methods:

• Site-directed mutagenesis of conserved glycine residues in the bacterial c(11) ring.
• Structural and biochemical analyses to determine c-subunit stoichiometry.
• Molecular dynamics simulations to analyze interface energetics and geometry.
• Spectroscopic interaction studies and real-time ATP synthesis experiments in proteoliposomes.

Main Results:

• Mutagenesis revealed a direct influence on c-subunit stoichiometry, generating rings with sizes less than 11, 12, 13, 14, and greater than 14 subunits.
• Molecular dynamics simulations provided insights into the energetic and geometric basis of these observed stoichiometries.
• Complex assembly was found to be independent of the c-ring size.
• Mutant ATP synthase with a larger c(12) ring demonstrated functionality at a lower ion motive force.

Conclusions:

• The flexibility in ATP synthase rotor architecture explains the natural variation in c-ring stoichiometries, likely an adaptation to specific bioenergetic needs.
• These findings pave the way for bioengineering ATP synthases with tailored ion-to-ATP ratios through sequence modifications.