Ligand-specific changes in conformational flexibility mediate long-range allostery in the lac repressor

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Protein allostery is crucial for biological regulation but poorly understood, particularly in proteins with subtle structural changes upon ligand binding.
• The lac repressor (LacI) is a model system for studying ligand-induced allostery.

Purpose of the Study:

• To investigate allosteric mechanisms in the lac repressor (LacI) using hydrogen-deuterium exchange with mass spectrometry (HDX/MS).
• To elucidate how ligand binding affects protein flexibility and conformational ensembles, even with minimal structural changes.

Main Methods:

• Utilized hydrogen-deuterium exchange with mass spectrometry (HDX/MS) to map protein flexibility.
• Analyzed LacI in various functional states: apo, inducer-bound, anti-inducer-bound, and DNA-bound.
• Integrated HDX/MS data with modeling of protein-ligand-solvent interactions.

Main Results:

• HDX/MS revealed significant differences in protein flexibility across different LacI functional states, despite near-identical crystal structures.
• Ligand binding was shown to allosterically shift the conformational ensemble by altering secondary structure rigidity.
• A revised model for LacI allostery was proposed, emphasizing flexibility changes over static structural alterations.

Conclusions:

• Ligand binding in LacI induces allostery through dynamic changes in protein flexibility, not just static structural shifts.
• This flexibility-based model explains the functional impact of distal mutations.
• The HDX/MS approach provides a versatile platform for studying and engineering protein allostery.