Figure 1.

Illustration of the “safety latch” hypothesis tested in this study. The crystallographic structure (32) of the T domain of diphtheria toxin (inset) is represented as the backbone ribbon with the consensus membrane-insertion domain, TH8–9, highlighted in brown. Histidines 223 (magenta) and 257 (purple) are shown as sticks. Solid lines represent MD-based calculations of the probability distribution that H257 has a particular pK_a (reproduced from (28)) (Two cases corresponding to the neighboring H223 being neutral (green trace) or charged (black trace) are considered. Because the histidine protonation is coupled with a pH-dependent conformational transition to form a membrane-competent state (discussed in detail in (29) and illustrated in Fig. S1), the conformational transition occurs at a pH corresponding to the rising edge of the pK_a distribution of the main trigger, H257 (25, 28) (vertical arrows). The protonation of H223 serves as a safety latch for the H257 trigger by moving the onset of pH-coupled refolding and insertion toward the endosomal pH range (gray band). This safety-latch hypothesis is tested here by comparing pH-dependent folding and membrane insertion of the WT T domain with that of an H223Q mutant that lacks the latch (see text for details). To see this figure in color, go online.