Membrane interactions of diphtheria toxin analyzed using in vitro synthesized mutants.

We have developed a system to study the interactions of diphtheria toxin with the cell surface using non‐toxic mutant proteins synthesized in vitro. Proteins obtained by N‐terminal deletions containing the whole B fragment bound strongly to cells. Deletions extending into the B fragment did not yield an autonomous binding domain. Loss of only the N‐terminal 3 kd of the B fragment significantly impaired the ability to recognize the receptor. This, together with previous reports that the C‐terminal end of the B fragment is required for binding, suggests that both ends of the B fragment are necessary for receptor recognition. Receptor bound diphtheria toxin undergoes a conformational change at pH less than 5.3 that results in translocation of the A fragment to the cytosol and the appearance of a B fragment‐derived 25 kd polypeptide (P25) resistant to externally applied protease. Only the B fragment was required for generation of P25. N‐terminal deletions of 130 amino acids or more resulted in proteins that gave rise to P25 at higher pH than full length toxin. Furthermore, a second protease‐inaccessible polypeptide of 18 kd (P18) was observed.