Model-guided mutagenesis drives functional studies of human NHA2, implicated in hypertension.
Human NHA2 is a poorly characterized Na(+)/H(+) antiporter recently implicated in essential hypertension. We used a range of computational tools and evolutionary conservation analysis to build and validate a three-dimensional model of NHA2 based on the crystal structure of a distantly related bacterial transporter, NhaA. The model guided mutagenic evaluation of transport function, ion selectivity, and pH dependence of NHA2 by phenotype screening in yeast. We describe a cluster of essential, highly conserved titratable residues located in an assembly region made of two discontinuous helices of inverted topology, each interrupted by an extended chain. Whereas in NhaA, oppositely charged residues compensate for partial dipoles generated within this assembly, in NHA2, polar but uncharged residues suffice. Our findings led to a model for transport mechanism that was compared to the well-known electroneutral NHE1 and electrogenic NhaA subtypes. This study establishes NHA2 as a prototype for the poorly understood, yet ubiquitous, CPA2 antiporter family recently recognized in plants and metazoans and illustrates a structure-driven approach to derive functional information on a newly discovered transporter.
Schushan
Maya
M
Department of Biochemistry, The George S Wise Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv, 69978 Tel-Aviv, Israel.
Xiang
Minghui
M
Bogomiakov
Pavel
P
Padan
Etana
E
Rao
Rajini
R
Ben-Tal
Nir
N
D000595:Amino Acid Sequence; D017920:Antiporters / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D027682:Cation Transport Proteins / Q000737:chemistry; D017124:Conserved Sequence; D029968:Escherichia coli Proteins / Q000737:chemistry; D019143:Evolution, Molecular; D006801:Humans; D006973:Hypertension / Q000209:etiology* / Q000235:genetics / Q000503:physiopathology; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry; D055672:Static Electricity; D040681:Structural Homology, Protein
D017920:Antiporters; D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; C102839:NhaA protein, E coli; D011994:Recombinant Proteins; C490998:SLC9A1 protein, human; C527589:SLC9B2 protein, human; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers