Crucial Role of H322 in Folding of the Diphtheria Toxin T-Domain into the Open-Channel State

Abstract

The translocation (T) domain plays a key role in the entry of diphtheria toxin into the cell. Upon endosomal acidification, the T-domain undergoes a series of conformational changes that lead to its membrane insertion and formation of a channel. Recently, we have reported that the triple replacement of the C-terminal histidines H322, H323 and H372 with glutamines prevents the formation of open channels in planar lipid bilayers. Here, we report that this effect is primarily due to the mutation of H322. We further examine the relationship between the loss of functionality and membrane folding in a series of mutants with C-terminal histidine substitutions using spectroscopic assays. The membrane insertion pathway for the mutants differs from that of the wild type as revealed by membrane-induced red-shift of tryptophan fluorescence at pH 6.0–6.5. T-domain mutants with replacements at H323 and H372, but not at H322, regain wild type-like spectroscopic signature upon further acidification. Circular dichroism measurements confirm that affected mutants misfold during insertion into vesicles. Conductance measurements reveal that substituting H322 dramatically reduces the numbers of properly folded channels in a planar bilayer, but the properties of the active channels appear to be unaltered. We propose that H322 plays an important role in the formation of open channels and is involved in guiding the proper insertion of the N-terminal region of the T-domain into the membrane.

Cellular Entry of Diphtheria Toxin Does Not Require Formation of the Open-Channel State by its Translocation Domain

“…Is it possible that the number of molecules in the OCS conformation is only a minor fraction of the entire population in model experiments? Our spectroscopic data indicate otherwise, suggesting a clear correlation between the ability of helix TH5 to insert in the OCS conformation or its precursor even in the absence of transbilayer potential (Figures 2 and 3 and [38]); 3. What is the mechanism of the translocation?…”

“…Because this changes the electrophoretic mobility of the tagged T-domain, N-terminus translocation can be detected and quantified through SDS-PAGE. In Figure 4a we show the relative translocation of the T-domain WT and various Cterminal single mutants at pH 5.8 plotted against previously determined OCS activity of the same mutants [38] (in order to allow a direct comparison with already published results, all proteins used in Figure 4 contain native W206). The measurements show that the three mutants maintain over 90% of the translocation activity despite the loss of channel activity, suggesting that replacing the OCSblocking mutations does not alter N-terminus's translocation.…”

“…In contrast, Pathway 1 indicates that the OCS is formed after the translocation, and passageway through the bilayer is formed via an unknown, possibly transient conformation. In order to distinguish between the two pathways, we will use new and published data to compare how various mutations affect the following measures of T-domain activity: (a) conductance in planar bilayers (i.e., formation of the OCS) [37,38,[45][46][47][48], (b) N-terminus translocation in vesicles [37,49], and ultimately (c) cell death assay based on inhibition of protein synthesis in vivo [37,46,48,50]. The starting structure on top corresponds to the crystal structure of the toxin at neutral pH [42], and consists of the C-domain (red), T-domain (helices color-coded according to OCS topology), and R-domain (green).…”

“…[37,46,48]. The data for H322Q, H323Q, and H372Q mutants (color-coded circles) are from Figure 4b and reference [38]. Blue and gray arrows indicate the expected correlation for the translocation occurring via Pathway 1 or 2, respectively (see Figure 1 and text for details).…”

“…As a result of these studies (Reviewed in [28]), we now understand that the insertion/refolding process occurs along a complex pathway, which includes multiple intermediates with staggered pH-dependent transitions [29][30][31][32][33]. A number of critical titratable residues involved in acid-induced conformational switching have been identified in a series of spectroscopic studies in vitro (e.g., H223 and H257 [32][33][34][35], E349 and D352 [36], and the C-terminal histidines H322, H323, and H372 [35,37,38]). Two recent studies of T-domain conductivity in planar lipid bilayers provided better understanding of the topological arrangements of the T-domain at the late stages of the pathway, at the so-called Open-Channel State (OCS) [39,40].…”