Structure and substrate ion binding in the sodium/proton antiporter PaNhaP.

Area of Science:

• Structural Biology
• Membrane Transport
• Biochemistry

Background:

• Sodium/proton antiporters are crucial for regulating intracellular pH and sodium ion concentrations.
• Understanding the precise structure of antiporters with bound ions is key to elucidating their transport mechanisms.
• The dimeric, electroneutral sodium/proton antiporter PaNhaP from Pyrococcus abyssi is a model system for studying these transporters.

Purpose of the Study:

• To determine the high-resolution structure of the PaNhaP antiporter with its bound substrate ion.
• To investigate the structural basis for pH-dependent conformational changes and transport activity.
• To elucidate the ion coordination and proton uptake pathways within the antiporter.

Main Methods:

• X-ray crystallography was used to resolve the structure of PaNhaP at 3.2 Å resolution.
• Structures were determined in two distinct conformations at pH 8 and pH 4.
• Transport assays were conducted to assess the cooperative activity of PaNhaP at different pH values.

Main Results:

• The structure reveals the bound sodium ion coordinated by acidic sidechains, a water molecule, a serine, and a main-chain carbonyl within a cytoplasmic funnel.
• A second polar channel potentially facilitates proton uptake.
• PaNhaP exhibits cooperative transport activity at pH 6, dependent on pH-induced allosteric coupling between protomers via histidines at the dimer interface, but not at pH 5.

Conclusions:

• The determined structures provide unprecedented detail on ion binding and coordination in sodium/proton antiporters.
• pH-dependent structural changes and allosteric coupling through specific residues explain the observed transport cooperativity.
• These findings offer significant new insights into the fundamental transport mechanism of sodium/proton antiporters.