# The crossing of two unwound transmembrane regions that is the hallmark of the NhaA structural fold is critical for antiporter activity

## TL;DR

The unique structure of NhaA, a protein that helps cells manage pH and sodium levels, is crucial for its function, and disrupting this structure blocks its activity.

## Contribution

The study shows that the X-shaped crossing of unwound transmembrane regions in NhaA is essential for its antiporter activity.

## Key findings

- Cross-linking at specific sites in NhaA inhibits antiporter activity and cell growth in high-salt conditions.
- The cross-linked NhaA protein is trapped in an outward-facing conformation, blocking its transport cycle.
- The NhaA structural fold is shared by other transporters, some of which are linked to human diseases.

## Abstract

Cell pH and Na+ homeostasis requires Na+/H+ antiporters. The crystal structure of NhaA, the main Escherichia coli Na+/H+ antiporter, revealed a unique NhaA structural fold shared by prokaryotic and eukaryotic membrane proteins. Out of the 12 NhaA transmembrane segments (TMs), TMs III–V and X–XII are topologically inverted repeats with unwound TMs IV and XI forming the X shape characterizing the NhaA fold. We show that intramolecular cross-linking under oxidizing conditions of a NhaA mutant with two Cys replacements across the crossing (D133C-T340C) inhibits antiporter activity and impairs NhaA-dependent cell growth in high-salts. The affinity purified D133C-T340C protein binds Li+ (the Na+ surrogate substrate of NhaA) under reducing conditions. The cross-linking traps the antiporter in an outward-facing conformation, blocking the antiport cycle. As many secondary transporters are found to share the NhaA fold, including some involved in human diseases, our data have importance for both basic and clinical research.