Kinetic Intermediate Reveals Staggered pH-Dependent Transitions along the Membrane Insertion Pathway of the Diphtheria Toxin T-Domain

Abstract

The pH-triggered membrane insertion pathway of the T-domain of diphtheria toxin was studied using site-selective fluorescence labeling with subsequent application of several spectroscopic techniques (e.g., fluorescence correlation spectroscopy, FRET, lifetime quenching and kinetic fluorescence). FCS measurements indicate that pH-dependent formation of the membrane-competent form depends only slightly on the amount of anionic lipids in the membrane. The subsequent transbilayer insertion, however, is strongly favored by anionic lipids. Kinetic FRET measurements between donor-labeled T-domain and acceptor-labeled lipid vesicles demonstrate rapid membrane association at all pH values for which binding occurs. In contrast, the transmembrane insertion kinetics is significantly slower, and is also both pH-and lipid-dependent. Analysis of kinetic behavior of binding and insertion indicates the presence of several interfacial intermediates on the insertion pathway of the T-domain, from soluble W-state to transmembrane T-state. Intermediate interfacial I-state can be trapped in membranes with low content of anionic lipids (10%). In membranes of greater anionic lipid content, another pH-dependent transition results in the formation of the insertion-competent state and subsequent transmembrane insertion. Comparison of the results of various kinetic and equilibrium experiments suggests that the pH-dependences determining membrane association and transbilayer insertion transitions are different, but staggered. Anionic lipids not only assist in formation of the insertion competent form, but also lower the kinetic barrier for the final insertion. The function of diphtheria toxin T-domain is to translocate the catalytic domain across the lipid bilayer in response to acidification of the endosome, a task this 178-residue protein is able to perform without the help of any other proteins (1). Although the exact mechanism of membrane translocation is not understood, protein refolding in the lipid bilayer environment has to be the central issue. Thus, deciphering the mechanism of pH-triggered DTT insertion is expected to impact not only the field of cellular entry of toxins, many of which also enter the cell via the *To whom correspondence should be addressed: Phone: 913-588-0489 FAX: 913-588-7440 aladokhin@kumc.edu. † This research was supported by NIH GM-069783 and GM-069783-S1. ‡ Permanent address for Drs. Kyrychenko and Posokhov is Institute for Chemistry at V.N. Karazin Kharkiv National University, 4 Svobody Sq., Kharkiv, 61077, Ukraine NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 August 18. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript endosomal pathway (2-4), but would also advance our understanding of general physicochemical principles underlying membrane protein assembly and stability.The crystallographic structure of DTT in the water-soluble form (5) (Fig. 1) provides a starting point for refolding/insertion studies. The protein consists of 9 ...

Tetanus Neurotoxin Utilizes Two Sequential Membrane Interactions for Channel Formation

“…Furthermore, CNTs do not undergo changes in secondary structure in response to low pH alone (Fig. 2), as has been reported for diphtheria toxin (42). Thus, the initial interaction of TeNT with the membrane does not appear to require the formation of a membrane-competent state in solution.…”

pH (low) insertion peptide (pHLIP) inserts across a lipid bilayer as a helix and exits by a different path

“…Several authors have reported peptide coil-helix transitions induced by binding to bilayers and the existence of interfacial folded intermediates, mostly using fluorescence approaches (19)(20)(21)(22)(23)(24). Although interesting, the results of molecular dynamics simulations of membrane-associated folding and insertion of various peptides are not definitive (25)(26)(27)(28).…”

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

“…On the right, the hydrophobic core of the T-domain is drawn in the OCS conformation with the three transmembrane helices, TH5, TH8, and TH9, and a dipped hairpin formed by shorter helices TH6 and TH7. This topology is based on a combination of measurements of channel activity in planar bilayers [39,40,43] and is supported by additional spectroscopic evidence in model lipid vesicles ( [30,44] and Kyrychenko et al, Journal of Membrane Biology, in press). The translocation Pathway 2 suggests that OCS is a functionally relevant state, which provides a passageway for translocation of the presumably unfolded catalytic domain to the other side of the bilayer (red circle-before translocation; pink circle-after translocation).…”

“…In pathway 2 (gray arrows), the T-domain first adopts the OCS conformation, which then serves as passageway for the translocation of the C-domain and the T-domain's N-terminal helices. The topology of the T-domain in the OCS, i.e., transmembrane helices TH5 (blue helix), and TH8-TH9 (purple helical hairpin), and interfacial helices TH6 and TH7 (gray helices), is based on conductance measurements on planar bilayers [39,40,43] and supported by spectroscopic data in lipid vesicles [30,44]. Replacement of the C-terminal histidines of the T-domain (H322Q, H323Q, H372Q) are known to strongly reduce formation of channels in planar bilayers by blocking the formation of the OCS conformation [37,38].…”

“…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]).…”