β-Sheet-dependent Dimerization Is Essential for the Stability of NhaA Na+/H+ Antiporter

Abstract

A structural model of the NhaA dimer showed that a ␤-hairpin of each monomer combines to form a ␤-sheet at the periplasmic side of the membrane. By Cys scanning the entire ␤-hairpin and testing each Cys replacement for functionality and intermolecular cross-linking, we found that Gln 47 and Arg 49 are critical for the NhaA dimer and that K57C causes an acidic shift of 1 pH unit to the pH dependence of NhaA. Comparing the growth of the NhaA variants with the previously isolated ␤-hairpin deleted mutant (⌬(P45-N58)) and the wild type validated that NhaA dimers have an advantage over monomers in growth under extreme stress conditions and unraveled that during this growth the apparent K m for Na ؉ of ⌬(P45-N58) was increased 50-fold as compared with the wild type. Remarkably, the effect of the extreme stress on the NhaA variants is reversible. Testing the temperature stability (4 -55°C) of the NhaA variants in dodecyl maltoside micells showed that the mutants impaired in dimerization were much less temperature-stable than the wild type. We suggest that NhaA dimers are crucial for the stability of the antiporter under extreme stress conditions.

NhaA antiporter functions using 10 helices, and an additional 2 contribute to assembly/stability

“…Thus, when a single amino acid in each NhaA monomer at the dimer interface is replaced with Cys ( Fig. 1A; V254C, S246C, or Q47C), that Cys residue cross-links with its twin in the other monomer (26,31). These Cys replacements were introduced into mutant Δ(VI-VII), and cross-linking was studied with bifunctional cross-linkers of different lengths, all of which yield similar results (Fig.…”

“…WT Ec-NhaA protein in DDM detergent micelles denatures at temperatures above 62.5°C (26) and, like many other membrane proteins, the protein aggregates at denaturing temperatures (Fig. 4B, Left).…”

“…The dimer is held together by interactions between the β-hairpins that form a β-sheet between the two monomers (25,26), and the crystal structure of the dimer shows (27) that TM VII may also contribute to interactions between monomers in the dimer interface. The reduced cooperativity of monomeric Ec-NhaA upon truncation (SI Text, Extended NMA Results) may also have indirect effects on dimerization.…”

“…WT EcNhaA is a stable dimer in the membrane (30,31) and in n-dodecyl-β-D-maltopyranoside (DDM) micelles (25,27), and the dimer interface has been identified by intermolecular cross-linking (26,31). Thus, when a single amino acid in each NhaA monomer at the dimer interface is replaced with Cys ( Fig.…”

Rotamer libraries of spin labelled cysteines for protein studies

“…5, orange) features a very small dimer interface. showing that deletions or mutations in that region can destabilize the dimer and impair viability of the bacteria under high-salt conditions 49 . The basic arrangement of the NhaA dimer was later independently confirmed by using the 3.45-Å X-ray structure of the NhaA monomer as a basis for molecular replacement in the 7-Å cryo-EM map acquired on 2D crystals 32 .…”