Structural and functional analysis of extracellular loop 2 of the Na(+)/H(+) exchanger.
The mammalian Na(+)/H(+) exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH (pHi) by removing one intracellular H(+) in exchange for one extracellular Na(+). It has a large 500 amino acid N-terminal membrane domain that mediates transport and consists of 12 transmembrane segments and several membrane-associated segments. Extracellular regions of this domain are believed to contribute to cation coordination, transport and sensitivity to inhibitors. In this study we characterized the structure and function of extracellular loop 2. Mutation of residues Pro153, Pro154 and Phe155 demonstrated that these residues were critical for efficient NHE1 function. Mutations to Ala resulted in decreases in cation affinity and in decreases in activity of the protein, these were more marked in both Pro154 and Phe155. NMR spectroscopy was used to characterize the solution structure of a peptide NAc-Gly150-Phe155-NH(2). The peptide showed at least three different conformers in solution due to cis-trans isomerization of the Thr(152)-Pro(153) and Pro(153)-Pro(154) peptide bonds. The trans-trans conformation appeared to be in an extended conformation, whereas the cis-trans conformation showed a propensity to form a beta turn. Our results show that the EL2 region is critical to NHE1 function and that a peptide of the EL2 region can adopt different structures in solution potentially forming a beta turn that is important in function of the full protein Mutation of Pro(154) could disrupt the beta turn, affecting helix packing and the protein structure and function.
Lee
Brian L
BL
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
Li
Xiuju
X
Liu
Yongsheng
Y
Sykes
Brian D
BD
Fliegel
Larry
L
D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D002414:Cations, Monovalent / Q000737:chemistry / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport / Q000502:physiology; D020125:Mutation, Missense; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000502:physiology; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship
D027682:Cation Transport Proteins; D002414:Cations, Monovalent; D010455:Peptides; C490998:SLC9A1 protein, human; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers