Structure-function analysis of the diphtheria toxin receptor toxin binding site by site-directed mutagenesis.
Diphtheria toxin (DT) binds to the epidermal growth factor (EGF)-like domain of human membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF), the human DT receptor (DTR). DT does not bind to mouse proHB-EGF because of amino acid substitutions within the EGF-like domain. We made 10 independent mutants, replacing a single amino acid within the EGF-like domain of human DTR/proHB-EGF with the corresponding amino acid residue in mouse proHB-EGF. The mutant proteins were transiently expressed in mouse L cells either expressing or not expressing DRAP27/CD9, and DT binding was measured. DT binding activity of GST fusion proteins containing the mutated EGF-like domain was also determined by a cell-free binding assay. The largest effect was seen with E141H, and second largest effects were seen with F115Y and L127F in all of the assay systems. We conclude that Phe115, Leu127, and Glu141 are critical amino acid residues for DT binding. A computer model of the tertiary structure of the EGF-like domain of human DTR/proHB-EGF was made. The model predicts that three amino acid residues critical for DT binding activity, Phe115, Leu127, and Glu141, are all located on the same face of the EGF-like domain, suggesting that this face of DTR/proHB-EGF interacts with the receptor-binding domain of DT.
Mitamura
T
T
Division of Cell Biology, Institute of Life Science, Kurume University, Kurume, Fukuoka 839 Japan.
Umata
T
T
Nakano
F
F
Shishido
Y
Y
Toyoda
T
T
Itai
A
A
Kimura
H
H
Mekada
E
E
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D003198:Computer Simulation; D004167:Diphtheria Toxin / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D066257:Heparin-binding EGF-like Growth Factor; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D004167:Diphtheria Toxin; C587883:HBEGF protein, human; C587884:Hbegf protein, mouse; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D006493:Heparin