Long-range coupling in an allosteric receptor revealed by mutant cycle analysis

Area of Science:

• Biochemistry
• Structural Biology
• Pharmacology

Background:

• Allosteric proteins exhibit functional coupling between distant residues.
• Cys-loop receptors, like nicotinic acetylcholine receptors, use allosteric regulation for ion channel gating.
• Identifying residues mediating communication between binding sites and gating regions (gating pathway residues) is challenging.

Purpose of the Study:

• To introduce a straightforward strategy for identifying gating pathway residues in allosteric receptors.
• To utilize macroscopic measurements and mutant cycle analysis for this identification.
• To elucidate long-range functional coupling in allosteric receptor systems.

Main Methods:

• Developed a method based on mutant cycle analysis using macroscopic measurements.
• Applied the method to the nicotinic acetylcholine receptor, a Cys-loop receptor.
• Combined a known reporter mutation (betaL9'S) with mutations in the ligand-binding domain.

Main Results:

• Mutant cycle analysis of EC(50) measurements provided insights into the roles of target residues.
• The method successfully identified functionally significant residues within the gating pathway.
• Macroscopic data interpretation was supported by single-channel studies.

Conclusions:

• The developed strategy effectively identifies gating pathway residues using macroscopic data alone.
• This approach elucidates long-range functional coupling in allosteric receptors.
• The method is broadly applicable to various receptors for identifying functionally important mutations.