Structure of the Diphtheria Toxin at Acidic pH: Implications for the Conformational Switching of the Translocation Domain
Diphtheria toxin, an exotoxin secreted by <i>Corynebacterium</i> that causes disease in humans by inhibiting protein synthesis, enters the cell via receptor-mediated endocytosis. The subsequent endosomal acidification triggers a series of conformational changes, resulting in the refoldin...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2020-11-01
|
| Series: | Toxins |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2072-6651/12/11/704 |
| _version_ | 1827702661507448832 |
|---|---|
| author | Mykola V. Rodnin Maithri M. Kashipathy Alexander Kyrychenko Kevin P. Battaile Scott Lovell Alexey S. Ladokhin |
| author_facet | Mykola V. Rodnin Maithri M. Kashipathy Alexander Kyrychenko Kevin P. Battaile Scott Lovell Alexey S. Ladokhin |
| author_sort | Mykola V. Rodnin |
| collection | DOAJ |
| description | Diphtheria toxin, an exotoxin secreted by <i>Corynebacterium</i> that causes disease in humans by inhibiting protein synthesis, enters the cell via receptor-mediated endocytosis. The subsequent endosomal acidification triggers a series of conformational changes, resulting in the refolding and membrane insertion of the translocation (T-)domain and ultimately leading to the translocation of the catalytic domain into the cytoplasm. Here, we use X-ray crystallography along with circular dichroism and fluorescence spectroscopy to gain insight into the mechanism of the early stages of pH-dependent conformational transition. For the first time, we present the high-resolution structure of the diphtheria toxin at a mildly acidic pH (5–6) and compare it to the structure at neutral pH (7). We demonstrate that neither catalytic nor receptor-binding domains change their structure upon this acidification, while the T-domain undergoes a conformational change that results in the unfolding of the TH2–3 helices. Surprisingly, the TH1 helix maintains its conformation in the crystal of the full-length toxin even at pH 5. This contrasts with the evidence from the new and previously published data, obtained by spectroscopic measurements and molecular dynamics computer simulations, which indicate the refolding of TH1 upon the acidification of the isolated T-domain. The overall results imply that the membrane interactions of the T-domain are critical in ensuring the proper conformational changes required for the preparation of the diphtheria toxin for the cellular entry. |
| first_indexed | 2024-03-10T15:01:43Z |
| format | Article |
| id | doaj.art-048109f7b54b413aa2201ff716c83348 |
| institution | Directory Open Access Journal |
| issn | 2072-6651 |
| language | English |
| last_indexed | 2024-03-10T15:01:43Z |
| publishDate | 2020-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Toxins |
| spelling | doaj.art-048109f7b54b413aa2201ff716c833482023-11-20T20:08:03ZengMDPI AGToxins2072-66512020-11-01121170410.3390/toxins12110704Structure of the Diphtheria Toxin at Acidic pH: Implications for the Conformational Switching of the Translocation DomainMykola V. Rodnin0Maithri M. Kashipathy1Alexander Kyrychenko2Kevin P. Battaile3Scott Lovell4Alexey S. Ladokhin5Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160, USAProtein Structure Laboratory, Shankel Structural Biology Center, University of Kansas, Lawrence, KS 66047, USADepartment of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160, USANYX beamline, New York Structural Biology Center, Upton, NY 11973, USAProtein Structure Laboratory, Shankel Structural Biology Center, University of Kansas, Lawrence, KS 66047, USADepartment of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160, USADiphtheria toxin, an exotoxin secreted by <i>Corynebacterium</i> that causes disease in humans by inhibiting protein synthesis, enters the cell via receptor-mediated endocytosis. The subsequent endosomal acidification triggers a series of conformational changes, resulting in the refolding and membrane insertion of the translocation (T-)domain and ultimately leading to the translocation of the catalytic domain into the cytoplasm. Here, we use X-ray crystallography along with circular dichroism and fluorescence spectroscopy to gain insight into the mechanism of the early stages of pH-dependent conformational transition. For the first time, we present the high-resolution structure of the diphtheria toxin at a mildly acidic pH (5–6) and compare it to the structure at neutral pH (7). We demonstrate that neither catalytic nor receptor-binding domains change their structure upon this acidification, while the T-domain undergoes a conformational change that results in the unfolding of the TH2–3 helices. Surprisingly, the TH1 helix maintains its conformation in the crystal of the full-length toxin even at pH 5. This contrasts with the evidence from the new and previously published data, obtained by spectroscopic measurements and molecular dynamics computer simulations, which indicate the refolding of TH1 upon the acidification of the isolated T-domain. The overall results imply that the membrane interactions of the T-domain are critical in ensuring the proper conformational changes required for the preparation of the diphtheria toxin for the cellular entry.https://www.mdpi.com/2072-6651/12/11/704diphtheria toxin structureX-ray crystallographyhelix unfoldingacidificationconformational switching |
| spellingShingle | Mykola V. Rodnin Maithri M. Kashipathy Alexander Kyrychenko Kevin P. Battaile Scott Lovell Alexey S. Ladokhin Structure of the Diphtheria Toxin at Acidic pH: Implications for the Conformational Switching of the Translocation Domain Toxins diphtheria toxin structure X-ray crystallography helix unfolding acidification conformational switching |
| title | Structure of the Diphtheria Toxin at Acidic pH: Implications for the Conformational Switching of the Translocation Domain |
| title_full | Structure of the Diphtheria Toxin at Acidic pH: Implications for the Conformational Switching of the Translocation Domain |
| title_fullStr | Structure of the Diphtheria Toxin at Acidic pH: Implications for the Conformational Switching of the Translocation Domain |
| title_full_unstemmed | Structure of the Diphtheria Toxin at Acidic pH: Implications for the Conformational Switching of the Translocation Domain |
| title_short | Structure of the Diphtheria Toxin at Acidic pH: Implications for the Conformational Switching of the Translocation Domain |
| title_sort | structure of the diphtheria toxin at acidic ph implications for the conformational switching of the translocation domain |
| topic | diphtheria toxin structure X-ray crystallography helix unfolding acidification conformational switching |
| url | https://www.mdpi.com/2072-6651/12/11/704 |
| work_keys_str_mv | AT mykolavrodnin structureofthediphtheriatoxinatacidicphimplicationsfortheconformationalswitchingofthetranslocationdomain AT maithrimkashipathy structureofthediphtheriatoxinatacidicphimplicationsfortheconformationalswitchingofthetranslocationdomain AT alexanderkyrychenko structureofthediphtheriatoxinatacidicphimplicationsfortheconformationalswitchingofthetranslocationdomain AT kevinpbattaile structureofthediphtheriatoxinatacidicphimplicationsfortheconformationalswitchingofthetranslocationdomain AT scottlovell structureofthediphtheriatoxinatacidicphimplicationsfortheconformationalswitchingofthetranslocationdomain AT alexeysladokhin structureofthediphtheriatoxinatacidicphimplicationsfortheconformationalswitchingofthetranslocationdomain |