Analysis of allosteric communication in a multienzyme complex by ancestral sequence reconstruction

Area of Science:

• Biochemistry and Molecular Evolution
• Protein Engineering and Bioinformatics

Background:

• Tryptophan synthase (TS) is a vital αββα heterotetrameric enzyme complex facilitating indole channeling.
• TS exhibits complex allosteric regulation between its α (TrpA) and β (TrpB) subunits, crucial for enzyme activity.
• Previous studies computationally resurrected the last bacterial common ancestor's (LBCA) TS, revealing differences in allosteric effects.

Purpose of the Study:

• To pinpoint specific amino acid residues responsible for the inversion of allosteric effects in TS evolution.
• To elucidate the evolutionary pathway of allosteric activation in the TrpA-TrpB interaction.
• To demonstrate the utility of ancestral sequence reconstruction (ASR) in identifying key residues in protein allostery.

Main Methods:

• Ancestral sequence reconstruction (ASR) to computationally generate 6 intermediate TS protein sequences.
• Biochemical characterization of these ancestral TS intermediates to analyze allosteric effects.
• Site-directed mutagenesis and molecular dynamics simulations to identify and rationalize critical residues.

Main Results:

• The switch from TrpA-mediated TrpB inhibition to activation occurred between two successive TS evolutionary intermediates.
• Four specific residues in TrpB were identified as crucial for allosteric activation by TrpA.
• Molecular dynamics simulations confirmed the role of identified residues in allosteric signal propagation.

Conclusions:

• ASR is a powerful tool for dissecting the evolution of protein function and allosteric mechanisms.
• The study identified specific residues that fundamentally altered the allosteric communication within the TS complex.
• Understanding these evolutionary changes provides insights into the design principles of enzyme regulation.