Sequence determinants of a transmembrane proton channel: an inverse relationship between stability and function

Area of Science:

• Biophysics
• Structural Biology
• Virology

Background:

• Integral membrane proteins fold within lipid bilayers, a process with debated driving forces.
• The M2 protein from influenza A virus is a model for transmembrane helix association.
• M2's proton channel is vital for virus function and targeted by amantadine.

Purpose of the Study:

• Investigate the energetic impact of mutations at M2 transmembrane helix interfaces.
• Determine how mutations affect M2TM tetramer stability and amantadine binding.
• Elucidate the relationship between M2 structure, stability, and function.

Main Methods:

• Analytical ultracentrifugation to assess oligomeric state and free energy of M2TM variants.
• Site-directed mutagenesis to create ten single-residue mutations (alanine or phenylalanine).
• Amantadine binding assays and structural modeling to analyze the drug-bound state.

Main Results:

• No mutations destabilized M2TM tetramerization, suggesting stability is sacrificed for function.
• All mutations either destabilized amantadine binding or were isoenergetic.
• Mutations altering side-chain volume significantly impacted amantadine-bound tetramer stability.
• A structural model of the amantadine-bound M2TM state was generated.

Conclusions:

• M2 protein stability is likely traded for functional flexibility, enabling rapid conformational changes for gating.
• Mutations can restrict conformational flexibility, potentially stabilizing specific states.
• Amantadine binding is sensitive to mutations at helix interfaces, providing insights into drug interaction and channel structure.