Single mutation in the A domain of diphtheria toxin results in a protein with altered membrane insertion behavior.
The insertion of the A domain of diphtheria toxin into model membranes has been shown to be both pH- and temperature-dependent (Hu and Holmes (1984) J. Biol. Chem. 259, 12226-12233). In this report, the insertion behavior of two mutant proteins of diphtheria toxin, CRM197 and CRM9, was studied and compared to that of wild-type toxin. Results indicated that both CRM197 and CRM9 resembled toxin with respect to the pH-dependence of binding to negatively-charged liposomes at room temperature. However, CRM197 differed from toxin with respect to both the pH- and temperature-dependence of fragment A insertion; fragment A197 inserts more readily into the bilayer at 0 degrees C and low pH or at neutral pH and room temperature than does wild type fragment A under these same conditions. This result indicates that the single amino acid substitution in the A domain of CRM197 facilitates entry of fragment A197 into the membrane, suggesting that CRM197 may be conformationally distinct from native toxin. In fact, the fluorescence spectra of CRM197 and wild-type toxin as well as their respective tryptic peptide patterns indicate that, at pH 7, CRM197 more closely resembles the acid form of wild-type toxin than the native form of toxin. These data suggest that CRM197 may be naturally in a more 'insertion-competent' conformation. In contrast, the mutation in the B domain of CRM9 which results in a 1000-fold decrease in binding affinity for plasma membrane receptors apparently does not cause a change in either the insertion of fragment A9 or the lipid-binding properties of CRM9 relative to toxin.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D004167:Diphtheria Toxin / Q000235:genetics / Q000378:metabolism*; D066257:Heparin-binding EGF-like Growth Factor; D006863:Hydrogen-Ion Concentration; D036341:Intercellular Signaling Peptides and Proteins; D008051:Lipid Bilayers / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism*; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014357:Trypsin
D001426:Bacterial Proteins; C053395:CRM9 protein, bacteria; D004167:Diphtheria Toxin; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D008051:Lipid Bilayers; D008081:Liposomes; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic; C028581:CRM197 (non-toxic variant of diphtheria toxin); D014357:Trypsin