Structure-function relationship of the fifth transmembrane domain in the Na+/H+ antiporter of Helicobacter pylori: Topology and function of the residues, including two consecutive essential aspartate residues.
We examined the structure-function relationships of residues in the fifth transmembrane domain (TM5) of the Na+/H+ antiporter A (NhaA) from Helicobacter pylori (HP NhaA) by cysteine scanning mutagenesis. TM5 contains two aspartate residues, Asp-171 and Asp-172, which are essential for antiporter activity. Thirty-five residues spanning the putative TM5 and adjacent loop regions were replaced by cysteines. Cysteines replacing Val-162, Ile-165, and Asp-172 were labeled with NEM, suggesting that these three residues are exposed to a hydrophilic cavity within the membrane. Other residues in the putative TM domain, including Asp-171, were not labeled. Inhibition of NEM labeling by the membrane impermeable reagent AMS suggests that Val-162 and Ile-165 are exposed to a water filled channel open to the cytoplasmic space, whereas Asp-172 is exposed to the periplasmic space. D171C and D172C mutants completely lost Na+/H+ and Li+/H+ antiporter activities, whereas other Cys replacements did not result in a significant loss of these activities. These results suggest that Asp-171 and Asp-172 and the surrounding residues of TM5 provide an essential structure for H+ binding and Na+ or Li+ exchange. A168C and Y183C showed markedly decreased antiporter activities at acidic pH, whereas their activities were higher at alkaline pH, suggesting that the conformation of TM5 also plays a crucial role in the HP NhaA-specific acidic pH antiporter activity.
Kuwabara
Naoyuki
N
Biological Sciences, Osaka University, Graduate School of Science, 1-16 Machikaneyama-cho 1-1, Toyonaka City, Osaka 560-0043, Japan.
Inoue
Hiroki
H
Tsuboi
Yumi
Y
Mitsui
Keiji
K
Matsushita
Masafumi
M
Kanazawa
Hiroshi
H
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003545:Cysteine; D016480:Helicobacter pylori / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D017923:Sodium-Hydrogen Exchangers; D003545:Cysteine