Ph-Triggered Membrane Insertion Pathway Of The Diphtheria Toxin T-Domain: 2. Role Of Histidines

The function of diphtheria toxin T-domain is to insert into the lipid bilayer and translocate the catalytic domain across the membrane in response to acidification of the endosome. In the accompanying presentation (Kyrychenko et al., BPS Meeting 2009) we have characterized the membrane insertion pathway of the T-domain WT. Here we examine the role of several crucial histidine residues in pH-dependent insertion by comparing WT to various mutants with one or more H-to-Q or H-to-R substitution(s). Thermal denaturation measured by CD indicates that all tested substitutions lead to lower stability of the protein. Kinetic measurements of guanidinum denaturation reveal that the folding branch of the chevron plot for the WT is independent of pH, while the unfolding branch becomes more prominent with acidification (a feature lost in some of the mutants). We have examined the insertion of the TH8-9 helical hairpin by following the topology of the NBD probe attached to C350, which is translocated across the bilayer in the WT. Interestingly, a single mutation of a remote histidine residue (H257R or H257Q) abolishes the insertion, while triple mutations of the H322, H323, H372, located on the top of the TH8-9 hairpin, do not affect the topology. This result is confirmed by emission measurements of the bimane probe placed in the middle of TH-9 helix: (1) WT-like blue shift for triple-R or triple-Q mutants and no shift for H257R or H257Q. The kinetics of membrane insertion, however, is affected in triple mutants: abolishing the charge (triple-Q mutant) results in slower insertion kinetics, while placing constant charges (triple-R mutant) in the same positions accelerates the insertion at intermediate pH. These results are consistent with the proposed role of His protonation in triggering membrane refolding. Supported by NIH GM069783(-04S1).