Cargando…

Apoptosome Formation through Disruption of the K192-D616 Salt Bridge in the Apaf-1 Closed Form

[Image: see text] The molecular mechanism of apoptosome activation through conformational changes of Apaf-1 auto-inhibited form remains largely enigmatic. The crystal structure of Apaf-1 suggests that some ionic bonds, including the bond between K192 and D616, are critical for the preservation of th...

Descripción completa

Detalles Bibliográficos
Autores principales: Sahebazzamani, Fatemeh, Hosseinkhani, Saman, Eriksson, Leif A., Fearnhead, Howard O.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2021
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8427654/
https://www.ncbi.nlm.nih.gov/pubmed/34514227
http://dx.doi.org/10.1021/acsomega.1c02274
_version_ 1783750220797968384
author Sahebazzamani, Fatemeh
Hosseinkhani, Saman
Eriksson, Leif A.
Fearnhead, Howard O.
author_facet Sahebazzamani, Fatemeh
Hosseinkhani, Saman
Eriksson, Leif A.
Fearnhead, Howard O.
author_sort Sahebazzamani, Fatemeh
collection PubMed
description [Image: see text] The molecular mechanism of apoptosome activation through conformational changes of Apaf-1 auto-inhibited form remains largely enigmatic. The crystal structure of Apaf-1 suggests that some ionic bonds, including the bond between K192 and D616, are critical for the preservation of the inactive “closed” form of Apaf-1. Here, a split luciferase complementation assay was used to monitor the effect of disrupting this ionic bond on apoptosome activation and caspase-3 activity in cells. The K192E mutation, predicted to disrupt the ionic interaction with D616, increased apoptosome formation and caspase activity, suggesting that this mutation favors the “open”/active form of Apaf-1. However, mutation of D616 to alanine or lysine had different effects. While both mutants favored apoptosome formation such as K192E, D616K cannot activate caspases and D616A activates caspases poorly, and not as well as wild-type Apaf-1. Thus, our data show that the ionic bond between K192 and D616 is critical for maintaining the closed form of Apaf-1 and that disrupting the interaction enhances apoptosome formation. However, our data also reveal that after apoptosome formation, D616 and K192 play a previously unsuspected role in caspase activation. The molecular explanation for this observation is yet to be elucidated.
format Online
Article
Text
id pubmed-8427654
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher American Chemical Society
record_format MEDLINE/PubMed
spelling pubmed-84276542021-09-10 Apoptosome Formation through Disruption of the K192-D616 Salt Bridge in the Apaf-1 Closed Form Sahebazzamani, Fatemeh Hosseinkhani, Saman Eriksson, Leif A. Fearnhead, Howard O. ACS Omega [Image: see text] The molecular mechanism of apoptosome activation through conformational changes of Apaf-1 auto-inhibited form remains largely enigmatic. The crystal structure of Apaf-1 suggests that some ionic bonds, including the bond between K192 and D616, are critical for the preservation of the inactive “closed” form of Apaf-1. Here, a split luciferase complementation assay was used to monitor the effect of disrupting this ionic bond on apoptosome activation and caspase-3 activity in cells. The K192E mutation, predicted to disrupt the ionic interaction with D616, increased apoptosome formation and caspase activity, suggesting that this mutation favors the “open”/active form of Apaf-1. However, mutation of D616 to alanine or lysine had different effects. While both mutants favored apoptosome formation such as K192E, D616K cannot activate caspases and D616A activates caspases poorly, and not as well as wild-type Apaf-1. Thus, our data show that the ionic bond between K192 and D616 is critical for maintaining the closed form of Apaf-1 and that disrupting the interaction enhances apoptosome formation. However, our data also reveal that after apoptosome formation, D616 and K192 play a previously unsuspected role in caspase activation. The molecular explanation for this observation is yet to be elucidated. American Chemical Society 2021-08-24 /pmc/articles/PMC8427654/ /pubmed/34514227 http://dx.doi.org/10.1021/acsomega.1c02274 Text en © 2021 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by-nc-nd/4.0/Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Sahebazzamani, Fatemeh
Hosseinkhani, Saman
Eriksson, Leif A.
Fearnhead, Howard O.
Apoptosome Formation through Disruption of the K192-D616 Salt Bridge in the Apaf-1 Closed Form
title Apoptosome Formation through Disruption of the K192-D616 Salt Bridge in the Apaf-1 Closed Form
title_full Apoptosome Formation through Disruption of the K192-D616 Salt Bridge in the Apaf-1 Closed Form
title_fullStr Apoptosome Formation through Disruption of the K192-D616 Salt Bridge in the Apaf-1 Closed Form
title_full_unstemmed Apoptosome Formation through Disruption of the K192-D616 Salt Bridge in the Apaf-1 Closed Form
title_short Apoptosome Formation through Disruption of the K192-D616 Salt Bridge in the Apaf-1 Closed Form
title_sort apoptosome formation through disruption of the k192-d616 salt bridge in the apaf-1 closed form
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8427654/
https://www.ncbi.nlm.nih.gov/pubmed/34514227
http://dx.doi.org/10.1021/acsomega.1c02274
work_keys_str_mv AT sahebazzamanifatemeh apoptosomeformationthroughdisruptionofthek192d616saltbridgeintheapaf1closedform
AT hosseinkhanisaman apoptosomeformationthroughdisruptionofthek192d616saltbridgeintheapaf1closedform
AT erikssonleifa apoptosomeformationthroughdisruptionofthek192d616saltbridgeintheapaf1closedform
AT fearnheadhowardo apoptosomeformationthroughdisruptionofthek192d616saltbridgeintheapaf1closedform