The NhaA Na⁺/H⁺ antiporter, new insights into structural and functional dynamics

Na⁺/H⁺ antiporters in the membranes of all cells are essential for homeostasis of intracellular pH, Na⁺, and volume. Solving the NhaA Na⁺/H⁺ antiporter crystal structure has been a breakthrough. The NhaA structural fold is unique: two inverted repeats of helices with unwound chain in each repeat cross each other forming a delicately elocrostatic balanced area in the middle of the membrane essential for activity. Remarkably, the number of secodary transporters with the NhaA fold is steadily increasing.

Transporter is a dynamic “nano-machine” which transports the substrate by alternating its access to either side of the membrane. We now crystallize active conformations of NhaA and employ interdiciplinary techniques to identify the pH/Li⁺/Na⁺ induced conformotianal changes. Employing Hydrogen-Deuterium Exchange Mass Spectroscopy evealed a global coordinated conformational change in the architecture of NhaA upon Li⁺ binding.

Comprehensive evolutionary analysis of the present 6,537 representative of Na⁺/H⁺ antiporter sequences describes the full complexity of the antiporters phylogeny as a tree, and reveals a sequence motif that determines the phenotypic characteristics of NhaA.

NhaA is a dimer with cardiolipin in the interface. We developed a novel straightforward in-vitro assay which likely be applicable to future studies of protein–lipid interactions: detergent delipidation split the dimers into monomers and subsequent exposure to cardiolipin reconstitute the dimers. In-vivo experiments and mutagenesis revealed the cardiolipin binding site and its importance in function. The human NhaA homologues involved in severe pathologies might also require specific phospholipids so our results open a new direction to drug design.