Epitope mapping of conformational monoclonal antibodies specific to NhaA Na+/H+ antiporter: structural and functional implications.
The recently determined crystal structure of NhaA, the Na(+)/H(+) antiporter of Escherichia coli, showed that the previously constructed series of NhaA-alkaline phosphatase (PhoA) fusions correctly predicted the topology of NhaA's 12 transmembrane segments (TMS), with the C- and N-termini pointing to the cytoplasm. Here, we show that these NhaA-PhoA fusions provide an excellent tool for mapping the epitopes of three NhaA-specific conformational monoclonal antibodies (mAbs), of which two drastically inhibit the antiporter. By identifying which of the NhaA fusions is bound by the respective mAb, the epitopes were localized to small stretches of NhaA. Then precise mapping was conducted by targeted Cys scanning mutagenesis combined with chemical modifications. Most interestingly, the epitopes of the inhibitory mAbs, 5H4 and 2C5, were identified in loop X-XI (cytoplasmic) and loop XI-XII (periplasmic), which are connected by TMS XI on the cytoplasmic and periplasmic sides of the membrane, respectively. The revealed location of the mAbs suggests that mAb binding distorts the unique NhaA TMS IV/XI assembly and thus inhibits the activity of NhaA. The noninhibitory mAb 6F9 binds to the functionally dispensable C-terminus of NhaA.
Rimon
Abraham
A
Department of Biochemistry, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
Hunte
Carola
C
Michel
Hartmut
H
Padan
Etana
E
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D018604:Epitope Mapping; D000939:Epitopes; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013267:Stilbenes / Q000737:chemistry; D013451:Sulfonic Acids / Q000737:chemistry
D000911:Antibodies, Monoclonal; D000939:Epitopes; D029968:Escherichia coli Proteins; C102839:NhaA protein, E coli; D011993:Recombinant Fusion Proteins; D017923:Sodium-Hydrogen Exchangers; D013267:Stilbenes; D013451:Sulfonic Acids; C079532:4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid; D000469:Alkaline Phosphatase; C114089:phoA protein, E coli; D003545:Cysteine