Active amino acids of the Kell blood group protein and model of the ectodomain based on the structure of neutral endopeptidase 24.11.
In addition to its importance in transfusion, Kell protein is a member of the M13 family of zinc endopeptidases and functions as an endothelin-3-converting enzyme. To obtain information on the structure of Kell protein we built a model based on the crystal structure of the ectodomain of neutral endopeptidase 24.11 (NEP). Similar to NEP, the Kell protein has 2 globular domains consisting mostly of alpha-helical segments. The domain situated closest to the membrane contains both the N- and C-terminal sequences and the enzyme-active site. The outer domain contains all of the amino acids whose substitutions lead to different Kell blood group phenotypes. In the model, the zinc peptidase inhibitor, phosphoramidon, was docked in the active site. Site-directed mutagenesis of amino acids in the active site was performed and the enzymatic activities of expressed mutant Kell proteins analyzed and compared with NEP. Our studies indicate that Kell and NEP use the same homologous amino acids in the coordination of zinc and in peptide hydrolysis. However, Kell uses different amino acids than NEP in substrate binding and appears to have more flexibility in the composition of amino acids allowed in the active site.
Lee
Soohee
S
Lindsley F. Kimball Research Institute, New York Blood Center, 310 E 67th St, New York, NY 10021, USA. solee@nybloodcenter.org
Debnath
Asim K
AK
Redman
Colvin M
CM
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D000954:Antigens, Surface / Q000737:chemistry* / Q000378:metabolism; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D002460:Cell Line; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D000072396:Endothelin-Converting Enzymes; D006020:Glycopeptides / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D007313:Insecta; D007626:Kell Blood-Group System; D008666:Metalloendopeptidases; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D015260:Neprilysin / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D015032:Zinc / Q000737:chemistry
D000596:Amino Acids; D000954:Antigens, Surface; D001798:Blood Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D006020:Glycopeptides; D007626:Kell Blood-Group System; C041106:kell-active proteins; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D015260:Neprilysin; D000072396:Endothelin-Converting Enzymes; D015032:Zinc; D003545:Cysteine; C008890:phosphoramidon