Histidine Protonation and Conformational Switching in Diphtheria Toxin Translocation Domain

Protonation of key histidine residues has been long implicated in the acid-mediated cellular action diphtheria toxin translocation (T-) domain, responsible for delivery catalytic domain into cell. Here, we use a combination computational (constant-pH Molecular Dynamics simulations) and experimental (NMR, circular dichroism, fluorescence spectroscopy along with X-ray crystallography) approaches to characterize initial stages conformational change happening solution wild-type T-domain H223Q/H257Q double mutant. This replacement suppresses acid-induced transition, resulting retention more stable protein structure solutions at pH 5.5 and, consequently, reduced membrane-disrupting activity. first time, report pKa values T-domain, measured by NMR-monitored titrations. Most peaks side chain spectral region are titrated pKas ranging from 6.2 6.8. However, two most up-field display little down 6, which is limiting this concentrations required NMR. These absent mutant, suggesting they belong H223 H257. The constant-pH simulations indicate that solution, range 3.0 6.5, those difficult protonate being H251 Taken together, our data demonstrate previously suggested cooperative protonation all six histidines does not occur.