Detection of Oligomerization and Conformational Changes in the Na+/H+ Antiporter from Helicobacter pylori by Fluorescence Resonance Energy Transfer

Akira Karasawa,Yumi Tsuboi,Hiroki Inoue,Rie Kinoshita,Norihiro Nakamura,Hiroshi Kanazawa

doi:10.1074/jbc.m510795200

Abstract

Oligomerization and conformational changes in the Na+/H+ antiporter from Helicobacter pylori (HPNhaA) were studied by means of fluorescence resonance energy transfer (FRET) analysis. Na+/H+ antiporter-deficient Escherichia coli cells expressing C-terminal fusions of HPNhaA to green fluorescent protein (GFP) variants exhibited wild-type levels of antiporter activity in their everted membrane vesicles. Vesicles containing both HPNhaA-CFP and HPNhaA-YFP or HPNhaA-Venus exhibited FRET from CFP (donor) to YFP or Venus (acceptor), suggesting that HPNhaA forms an oligomer. Co-precipitation of HPNhaA tagged by Venus and FLAG sequences confirmed oligomerization. FRET decreased extensively after treatment of the vesicles with proteinase K, which released GFP variants from the fusion proteins. FRET was not observed by merely mixing vesicles expressing the donor or acceptor fusion alone. Fluorescence of Venus is less sensitive to anions and stronger than that of anion-sensitive YFP. Using HPNhaA-Venus as the acceptor, Li+ was found to cause a significant decrease in FRET regardless of the presence or absence of DeltapH across the membranes, whereas Na+ caused a much weaker effect. This Li+ effect was minimal in vesicles prepared from cells expressing HPNhaA containing an Asp141 to Asn mutation, which results in defective Li+/H+ antiporter activity, possibly Li+ binding. These results demonstrate that monomer interactions within the HPNhaA oligomer are weakened possibly by Li+ binding. Dynamic interactions between HPNhaA monomers were detectable in membranes by FRET analysis, thus providing a new approach to study dynamic conformational changes in NhaA during antiport activity.

Highlights

We have shown for H. pylori NhaA (HPNhaA) that the enhancement of activity at alkaline pH is associated with Loop 7 and TM8, whereas the high activity at acidic pH is caused by a structure formed by TM4 and TM10 [12]
We have shown previously that the N- and C-terminal halves of HPNhaA interact functionally with each other based on the analysis of a chimeric NhaA construct of the E. coli and H. pylori proteins [15]
Whereas cell growth was vigorous for cells transformed with the C-terminal green fluorescent protein (GFP) fusion (TABLE ONE), the cell growth in high salt medium was very low for fusions with GFP variants at both their N and C termini (TABLE ONE)

Summary

Introduction

Three Asp residues essential for ion transport, as well as several other functionally important residues, have been identified for ECNhaA [11] and H. pylori NhaA (HPNhaA) [12]. These residues are clustered in transmembrane domains (TM) 4, 5, 10, and 11 in both HPNhaA [12] and ECNhaA [11]. Several primary missense mutations defective in antiport activity have been identified in these TMs, and suppressor mutations were mapped to these same TMs and flanking regions, suggesting mutual association of these TMs [12] This was directly shown by cross-linking of ECNhaA TM4 and TM11 [13]. Membrane vesicles (100 ␮g) were incubated in 2 ml of assay buffer, and pH-dependent antiporter activity was measured as the fluorescence change of ACMA in the presence of a ⌬pH across the membrane driven by 5 mM lactate, 5 mM

Methods

Results

Conclusion