Critical Functions of Region 1-67 and Helix XIII in Retaining the Active Structure of NhaD Antiporter in Halomonas sp. Y2

Frontiers in Microbiology
Zhou YangChunyu Yang

Abstract

NhaD-type antiporters are mainly distributed in various Proteobacteria, especially in marine microorganisms and human pathogens. This distribution as well as the pathogenic properties of these strains suggest that these antiporters contribute to the regulation of high osmoregulation and are potential drug targets. Two NhaD homologs, NhaD1 and NhaD2, from the halotolerant and alkaliphilic Halomonas sp. Y2 exhibits similar, high in vitro activity, but remarkably different in vivo functions. To search for critical domains or residues involved in these differences of physiological functions, various chimeras composed of NhaD1 and NhaD2 segments were generated. Two regions at residues 1-67 and 464-492 were found to be responsible for the robust in vivo function of NhaD2, and region 464-492 is also crucial to the pH response of the antiporter. In particular, the completely abolished activity of KNabc/N463r, highly recovered activity while very weakly recovered ion resistance of the KNabc/N463r-C7 chimera, suggested that transmembrane helix (TM) XIII is crucial for the robust ion resistance of NhaD2. Using site-directed mutagenesis, seven hydrophobic residues in TM XIII were identified as key residues for the ion translocation of NhaD...Continue Reading

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Methods Mentioned

BETA
environmental stress
PCR
protein assay
Reverse Transcription PCR
fluorescence resonance
FRET
Fluorescence

Software Mentioned

blunt
BlastP
pEASY
Clustal

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