Structural basis for Na(+) transport mechanism by a light-driven Na(+) pump

Area of Science:

• Biophysics
• Optogenetics
• Structural Biology

Background:

• Krokinobacter eikastus rhodopsin 2 (KR2) is the first identified light-driven Na(+) pump.
• Its mechanism for transporting non-proton cations was previously unknown due to the Schiff base proton.
• KR2 is a promising tool for next-generation optogenetics.

Purpose of the Study:

• To elucidate the molecular mechanism of Na(+) transport in KR2.
• To understand the gating mechanism enabling non-proton cation transport.
• To provide a framework for developing advanced optogenetics tools.

Main Methods:

• X-ray crystallography to determine KR2 structures in resting and M-like states.
• Spectroscopic analyses to investigate the gating mechanism.
• Structure-based engineering of light-driven K(+) pumps.
• Electrophysiological and behavioral assays in neurons and nematodes.

Main Results:

• Crystal structures revealed the resting and M-like intermediate states of KR2.
• The flipping of Asp116 was identified as the key gating mechanism, sequestering the Schiff base proton.
• This mechanism facilitates Na(+) transport through the ion-conducting pathway.
• Engineered K(+) pumps and functional assays confirmed the mechanism's role in non-proton cation transport.

Conclusions:

• The molecular basis for light-driven non-proton cation pumps, like KR2, has been revealed.
• The Asp116 gating mechanism is crucial for sequestering the Schiff base proton and enabling Na(+) transport.
• These findings provide a foundation for advancing the development of next-generation optogenetics tools.