Topography of Helices 5–7 in Membrane-inserted Diphtheria Toxin T Domain: IDENTIFICATION AND INSERTION BOUNDARIES OF TWO HYDROPHOBIC SEQUENCES THAT DO NOT FORM A STABLE TRANSMEMBRANE HAIRPIN

Michael P Rosconi,Erwin London

doi:10.1074/jbc.m200442200

Abstract

The T domain of diphtheria toxin undergoes a low pH-induced conformational change that allows it to penetrate cell membranes. T domain hydrophobic helices 8 and 9 can adopt two conformations, one close to the membrane surface (P state) and a second in which they apparently form a transmembrane hairpin (TM state). We have now studied T domain helices 5-7, a second cluster of hydrophobic helices, using Cys-scanning mutagenesis. After fluorescently labeling a series of Cys residues, penetration into a non-polar environment, accessibility to externally added antibodies, and relative depth in the bilayer were monitored. It was found that helices 5-7 insert shallowly in the P state and deeply in the TM state. Thus, the conformational changes in helices 5-7 are both similar and somehow linked to those in helices 8 and 9. The boundaries of deeply inserting sequences were also identified. One deeply inserted segment was found to span residues 270 to 290, which overlaps helix 5, and a second spanned residues 300 to 320, which includes most of helix 6 and all of helix 7. This indicates that helices 6 and 7 form a continuous hydrophobic segment despite their separation by a Pro-containing kink. Additionally, it is found that in the TM state some residues in the hydrophilic loop between helices 5 and 6 become more highly exposed than they are in the P state. Their exposure to external solution in the TM state indicates that helices 5-7 do not form a stable transmembrane hairpin. However, helix 5 and/or helices 6 plus 7 could form transmembrane structures that are in equilibrium with non-transmembrane states, or be kinetically prevented from forming a transmembrane structure. How helices 5-7 might influence the mechanism by which the T domain aids translocation of the diphtheria toxin A chain across membranes is discussed.

Highlights

The T domain of diphtheria toxin undergoes a low pH-induced conformational change that allows it to penetrate cell membranes
Cys-scanning/Fluorescence Labeling Strategy—In previous studies, we have shown that the T domain in the shallowly inserted (P) and deeply inserted (TM) conformations can be distinguished by measuring the fluorescence of membraneinserted T domains containing single-Cys residues labeled with bimane or BODIPY [18, 19]
Bimane and BODIPY labeling was extended to the hydrophobic helix 5–7 region of the T domain and adjacent sequences (Fig. 1)

Summary

Introduction

The T domain of diphtheria toxin undergoes a low pH-induced conformational change that allows it to penetrate cell membranes. Under conditions in which the membrane-inserted T domain forms the P conformation, bimane emission is relatively red-shifted throughout the helix 5–7 region (Fig. 3A).

Results

Conclusion