pH-dependent conformational changes in the HCV NS3 protein modulate its ATPase and helicase activities.
The hepatitis C virus (HCV) infects 170 to 200 million people worldwide and is, therefore, a major health problem. The lack of efficient treatments that specifically target the viral proteins or RNA and its high chronicity rate make hepatitis C the cause of many deaths and hepatic transplants annual...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2014-01-01
|
| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4281115?pdf=render |
| _version_ | 1818457256509833216 |
|---|---|
| author | Gustavo Tavares Ventura Emmerson Corrêa Brasil da Costa Anne Miranda Capaccia Ronaldo Mohana-Borges |
| author_facet | Gustavo Tavares Ventura Emmerson Corrêa Brasil da Costa Anne Miranda Capaccia Ronaldo Mohana-Borges |
| author_sort | Gustavo Tavares Ventura |
| collection | DOAJ |
| description | The hepatitis C virus (HCV) infects 170 to 200 million people worldwide and is, therefore, a major health problem. The lack of efficient treatments that specifically target the viral proteins or RNA and its high chronicity rate make hepatitis C the cause of many deaths and hepatic transplants annually. The NS3 protein is considered an important target for the development of anti-HCV drugs because it is composed of two domains (a serine protease in the N-terminal portion and an RNA helicase/NTPase in the C-terminal portion), which are essential for viral replication and proliferation. We expressed and purified both the NS3 helicase domain (NS3hel) and the full-length NS3 protein (NS3FL) and characterized pH-dependent structural changes associated with the increase in their ATPase and helicase activities at acidic pH. Using intrinsic fluorescence experiments, we have observed that NS3hel was less stable at pH 6.4 than at pH 7.2. Moreover, binding curves using an extrinsic fluorescent probe (bis-ANS) and ATPase assays performed under different pH conditions demonstrated that the hydrophobic clefts of NS3 are significantly more exposed to the aqueous medium at acidic pH. Using fluorescence spectroscopy and anisotropy assays, we have also observed more protein interaction with DNA upon pH acidification, which suggests that the hydrophobic clefts exposure on NS3 might be related to a loss of stability that could lead it to adopt a more open conformation. This conformational change at acidic pH would stimulate both its ATPase and helicase activities, as well as its ability to bind DNA. Taken together, our results indicate that the NS3 protein adopts a more open conformation due to acidification from pH 7.2 to 6.4, resulting in a more active form at a pH that is found near Golgi-derived membranes. This increased activity could better allow NS3 to carry out its functions during HCV replication. |
| first_indexed | 2024-12-14T22:39:40Z |
| format | Article |
| id | doaj.art-7173f1c01e124b85a789d8394322f904 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-14T22:39:40Z |
| publishDate | 2014-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-7173f1c01e124b85a789d8394322f9042022-12-21T22:45:00ZengPublic Library of Science (PLoS)PLoS ONE1932-62032014-01-01912e11594110.1371/journal.pone.0115941pH-dependent conformational changes in the HCV NS3 protein modulate its ATPase and helicase activities.Gustavo Tavares VenturaEmmerson Corrêa Brasil da CostaAnne Miranda CapacciaRonaldo Mohana-BorgesThe hepatitis C virus (HCV) infects 170 to 200 million people worldwide and is, therefore, a major health problem. The lack of efficient treatments that specifically target the viral proteins or RNA and its high chronicity rate make hepatitis C the cause of many deaths and hepatic transplants annually. The NS3 protein is considered an important target for the development of anti-HCV drugs because it is composed of two domains (a serine protease in the N-terminal portion and an RNA helicase/NTPase in the C-terminal portion), which are essential for viral replication and proliferation. We expressed and purified both the NS3 helicase domain (NS3hel) and the full-length NS3 protein (NS3FL) and characterized pH-dependent structural changes associated with the increase in their ATPase and helicase activities at acidic pH. Using intrinsic fluorescence experiments, we have observed that NS3hel was less stable at pH 6.4 than at pH 7.2. Moreover, binding curves using an extrinsic fluorescent probe (bis-ANS) and ATPase assays performed under different pH conditions demonstrated that the hydrophobic clefts of NS3 are significantly more exposed to the aqueous medium at acidic pH. Using fluorescence spectroscopy and anisotropy assays, we have also observed more protein interaction with DNA upon pH acidification, which suggests that the hydrophobic clefts exposure on NS3 might be related to a loss of stability that could lead it to adopt a more open conformation. This conformational change at acidic pH would stimulate both its ATPase and helicase activities, as well as its ability to bind DNA. Taken together, our results indicate that the NS3 protein adopts a more open conformation due to acidification from pH 7.2 to 6.4, resulting in a more active form at a pH that is found near Golgi-derived membranes. This increased activity could better allow NS3 to carry out its functions during HCV replication.http://europepmc.org/articles/PMC4281115?pdf=render |
| spellingShingle | Gustavo Tavares Ventura Emmerson Corrêa Brasil da Costa Anne Miranda Capaccia Ronaldo Mohana-Borges pH-dependent conformational changes in the HCV NS3 protein modulate its ATPase and helicase activities. PLoS ONE |
| title | pH-dependent conformational changes in the HCV NS3 protein modulate its ATPase and helicase activities. |
| title_full | pH-dependent conformational changes in the HCV NS3 protein modulate its ATPase and helicase activities. |
| title_fullStr | pH-dependent conformational changes in the HCV NS3 protein modulate its ATPase and helicase activities. |
| title_full_unstemmed | pH-dependent conformational changes in the HCV NS3 protein modulate its ATPase and helicase activities. |
| title_short | pH-dependent conformational changes in the HCV NS3 protein modulate its ATPase and helicase activities. |
| title_sort | ph dependent conformational changes in the hcv ns3 protein modulate its atpase and helicase activities |
| url | http://europepmc.org/articles/PMC4281115?pdf=render |
| work_keys_str_mv | AT gustavotavaresventura phdependentconformationalchangesinthehcvns3proteinmodulateitsatpaseandhelicaseactivities AT emmersoncorreabrasildacosta phdependentconformationalchangesinthehcvns3proteinmodulateitsatpaseandhelicaseactivities AT annemirandacapaccia phdependentconformationalchangesinthehcvns3proteinmodulateitsatpaseandhelicaseactivities AT ronaldomohanaborges phdependentconformationalchangesinthehcvns3proteinmodulateitsatpaseandhelicaseactivities |