Oligomerization of membrane-bound diphtheria toxin (CRM197) facilitates a transition to the open form and deep insertion.
Diphtheria toxin (DT) contains separate domains for receptor-specific binding, translocation, and enzymatic activity. After binding to cells, DT is taken up into endosome-like acidic compartments where the translocation domain inserts into the endosomal membrane and releases the catalytic domain into the cytosol. The process by which the catalytic domain is translocated across the endosomal membrane is known to involve pH-induced conformational changes; however, the molecular mechanisms are not yet understood, in large part due to the challenge of probing the conformation of the membrane-bound protein. In this work neutron reflection provided detailed conformational information for membrane-bound DT (CRM197) in situ. The data revealed that the bound toxin oligomerizes with increasing DT concentration and that the oligomeric form (and only the oligomeric form) undergoes a large extension into solution with decreasing pH that coincides with deep insertion of residues into the membrane. We interpret the large extension as a transition to the open form. These results thus indicate that as a function of bulk DT concentration, adsorbed DT passes from an inactive state with a monomeric dimension normal to the plane of the membrane to an active state with a dimeric dimension normal to the plane of the membrane.
Sandia National Laboratories, Albuquerque, New Mexico, USA. mskent@sandia.gov
M S
MS
Kent
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H
Yim
J K
JK
Murton
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Satija
J
J
Majewski
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I
Kuzmenko
D000327:Adsorption; D001426:Bacterial Proteins / Q000737:chemistry*; D001703:Biophysics / Q000379:methods; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004167:Diphtheria Toxin / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008055:Lipids / Q000737:chemistry; D021962:Membrane Microdomains / Q000737:chemistry; D015233:Models, Statistical; D008968:Molecular Conformation; D009502:Neutrons; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D004167:Diphtheria Toxin; D008055:Lipids; C028581:CRM197 (non-toxic variant of diphtheria toxin)