Accessibility changes within diphtheria toxin T domain upon membrane penetration probed by hydrogen exchange and mass spectrometry

 

Petr Man1,2, Caroline Montagner3, Heidi Vitrac3, Daniel Kavan1,2, Sylvain Pichard4, Daniel Gillet4, Eric Forest5 and Vincent Forge3

 

1Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic

2Department of Biochemistry, Faculty of Science, Charles University in Prague, Prague, Czech Republic

3 Institut de Recherches en Technologies et Sciences pour le Vivant, CEA, Grenoble, France

4Institut de Biologie et Technologies de Saclay, CEA, Gif sur Yvette, France

5 Institut de Biologie Structurale, Grenoble, France

 

Diphtheria toxin is a protein secreted by Corynebacterium diphtheriae as a single polypeptide chain of 58 kDa. During cell intoxication, it is cleaved by furin into two fragments, the A chain corresponding to the catalytic (C) domain and the B chain corresponding to the translocation (T) and receptor-binding domains. The C and T domains remain covalently linked by a disulfide bond. Following binding to its cell surface receptor, diphtheria toxin is internalized through the clathrin coated pathway. The acidic pH in the endosome triggers a conformational change leading to the insertion of the toxin in the membrane. The C domain is then translocated across the endosomal membrane into the cytosol. The C domain ADP-ribosylates the elongation factor 2, blocking protein translation and leading to cell death.

At neutral pH, the T domain is a globular protein made of ten alpha-helices, named TH1 to TH9 and TH5'. The insertion of the T domain into membranes is pH-dependent and follows a two-step process. During the first step, between pH 7 and pH 6, the T domain adopts a partially folded state, the so-called molten globule, because of the tertiary structure destabilization. At that stage, the T domain binds to membrane, mainly through hydrophobic interactions, leading to the membrane-bound (MB) state. During the second step, between pH 6 and pH 4, the conformation of the T domain is reorganized, leading to a membrane-inserted (MI) state. While the tertiary structure is lost, the secondary structure, i.e. the helical content, of the T domain is preserved throughout the process. The MI state is the functional state of the T domain; it permeabilizes the membrane and enables the passage of the N-terminal region from the cis to the trans side of the membrane.

In this work we used Hydrogen/Deuterium exchange coupled to mass spectrometry to describe the individual steps of T-domain membrane insertion. Accessibility changes of T-domain were followed for T-domain at pH 7, 6 and 4 and for different states either free T-domain in solution, or T-domain bound to the lipid membrane (mimicked by large unilamellar vesicles). Also the influence of high-ionic strength (masking of electrostatic interactions) was followed.

 

Financial support from CEA, MSMT LC 545 and Institutional research concept AV0Z50200510 is gratefully acknowledged.