Replacement of C-terminal histidines uncouples membrane insertion and translocation in diphtheria toxin T-domain

Mykola V. Rodnin, Alexander Kyrychenko, Paul Kienker, Onkar Sharma, Mauricio Vargas-Uribe, R. John Collier, Alan Finkelstein, Alexey S. Ladokhin

Research output: Contribution to journalArticle

21 Scopus citations

Abstract

The translocation (T) domain plays a key role in the action of diphtheria toxin and is responsible for transferring the N-terminus-attached catalytic domain across the endosomal membrane into the cytosol in response to acidification. The T-domain undergoes a series of pH-triggered conformational changes that take place in solution and on the membrane interface, and ultimately result in transbilayer insertion and N-terminus translocation. Structure-function studies along this pathway have been hindered because the protein population occupies multiple conformations at the same time. Here we report that replacement of the three C-terminal histidine residues, H322, H323, and H372, in triple-R or triple-Q mutants prevents effective translocation of the N-terminus. Introduction of these mutations in the full-length toxin results in decrease of its potency. In the context of isolated T-domain, these mutations cause loss of characteristic conductance in planar bilayers. Surprisingly, these mutations do not affect general folding in solution, protein interaction with the membranes, insertion of the consensus transmembrane helical hairpin TH8-9, or the ability of the T-domain to destabilize vesicles to cause leakage of fluorescent markers. Thus, the C-terminal histidine residues are critical for the transition from the inserted intermediate state to the open-channel state in the insertion/translocation pathway of the T-domain.

Original languageEnglish (US)
Pages (from-to)L41-L43
JournalBiophysical journal
Volume101
Issue number10
DOIs
StatePublished - Nov 16 2011

ASJC Scopus subject areas

  • Biophysics

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    Rodnin, M. V., Kyrychenko, A., Kienker, P., Sharma, O., Vargas-Uribe, M., Collier, R. J., Finkelstein, A., & Ladokhin, A. S. (2011). Replacement of C-terminal histidines uncouples membrane insertion and translocation in diphtheria toxin T-domain. Biophysical journal, 101(10), L41-L43. https://doi.org/10.1016/j.bpj.2011.10.018