Abstract
The translocation (T)-domain plays a key role in the action of diphtheria toxin and is responsible for transferring the catalytic domain across the endosomal membrane into the cytosol in response to acidification. Deciphering the molecular mechanism of pH-dependent refolding and membrane insertion of the T-domain, which is considered to be a paradigm for cell entry of other bacterial toxins, reveals general physicochemical principles underlying membrane protein assembly and signaling on membrane interfaces. Structure-function studies along the T-domain insertion pathway have been affected by the presence of multiple conformations at the same time, which hinders the application of high-resolution structural techniques. Here, we review recent progress in structural, functional and thermodynamic studies of the T-domain archived using a combination of site-selective fluorescence labeling with an array of spectroscopic techniques and computer simulations. We also discuss the principles of conformational switching along the insertion pathway revealed by studies of a series of T-domain mutants with substitutions of histidine residues.
Highlights
Diphtheria toxin enters the cell via the endosomal pathway [1], which is shared by many other toxins, including botulinum, tetanus and anthrax [2,3,4,5]
The protein consists of nine helices of various lengths (TH1-9), eight of which completely surround the most hydrophobic one, TH8
Direct observation of an interfacially refolded kinetic intermediate in the T-domain insertion pathway confirms the importance of understanding the various physicochemical phenomena that occur on membrane interfaces
Summary
Diphtheria toxin enters the cell via the endosomal pathway [1], which is shared by many other toxins, including botulinum, tetanus and anthrax [2,3,4,5]. Toxins 2013, 5 of these toxins are complex and not fully understood It is clear, that they have certain similarities with the entry pathway of diphtheria toxin: they involve receptor-mediated endocytosis followed by endosome acidification and pH-triggered conformational change that results in membrane insertion of the transporting protein and the formation of a pore or a transient passageway through which the toxic enzymatic components enter the cell (Figure 1). We will review the results of structural and thermodynamic studies of T-domain refolding and membrane insertion obtained in our lab for the past decade. T-domain resulting in refolding, membrane insertion and translocation of the C-domain (highlighted by the red rectangle)
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