Exploring the CNOT1(800–999) HEAT Domain and Its Interactions with Tristetraprolin (TTP) as Revealed by Hydrogen/Deuterium Exchange Mass Spectrometry
CNOT1, a key scaffold in the CCR4-NOT complex, plays a critical role in mRNA decay, particularly in the regulation of inflammatory responses through its interaction with tristetraprolin. A fragment of the middle part of CNOT1 (residues 800–999) is an example of an α-helical HEAT-like repeat domain....
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MDPI AG
2025-03-01
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| author | Maja K. Cieplak-Rotowska Michał Dadlez Anna Niedzwiecka |
| author_facet | Maja K. Cieplak-Rotowska Michał Dadlez Anna Niedzwiecka |
| author_sort | Maja K. Cieplak-Rotowska |
| collection | DOAJ |
| description | CNOT1, a key scaffold in the CCR4-NOT complex, plays a critical role in mRNA decay, particularly in the regulation of inflammatory responses through its interaction with tristetraprolin. A fragment of the middle part of CNOT1 (residues 800–999) is an example of an α-helical HEAT-like repeat domain. The HEAT motif is an evolutionarily conserved motif present in scaffolding and transport proteins across a wide range of organisms. Using hydrogen/deuterium exchange mass spectrometry (HDX MS), a method that has not been widely explored in the context of HEAT repeats, we analysed the structural dynamics of wild-type CNOT1(800–999) and its two double point mutants (E893A/Y900A, E893Q/Y900H) to find the individual contributions of these CNOT1 residues to the molecular recognition of tristetraprolin (TTP). Our results show that the differences in the interactions of CNOT1(800–999) variants with the TTP peptide fragment are due to the absence of the critical residues resulting from point mutations and not due to the perturbation of the protein structure. Nevertheless, the HDX MS was able to detect slight local changes in structural dynamics induced by protein point mutations, which are usually neglected in studies of intermolecular interactions. |
| format | Article |
| id | doaj-art-b890ee7aeace4bc3962f1c141f6319c0 |
| institution | OA Journals |
| issn | 2218-273X |
| language | English |
| publishDate | 2025-03-01 |
| publisher | MDPI AG |
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| series | Biomolecules |
| spelling | doaj-art-b890ee7aeace4bc3962f1c141f6319c02025-08-20T02:11:22ZengMDPI AGBiomolecules2218-273X2025-03-0115340310.3390/biom15030403Exploring the CNOT1(800–999) HEAT Domain and Its Interactions with Tristetraprolin (TTP) as Revealed by Hydrogen/Deuterium Exchange Mass SpectrometryMaja K. Cieplak-Rotowska0Michał Dadlez1Anna Niedzwiecka2Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, PL-02089 Warsaw, PolandLaboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, PL-02106 Warsaw, PolandLaboratory of Biological Physics, Institute of Physics, Polish Academy of Sciences, Aleja Lotnikow 32/46, PL-02668 Warsaw, PolandCNOT1, a key scaffold in the CCR4-NOT complex, plays a critical role in mRNA decay, particularly in the regulation of inflammatory responses through its interaction with tristetraprolin. A fragment of the middle part of CNOT1 (residues 800–999) is an example of an α-helical HEAT-like repeat domain. The HEAT motif is an evolutionarily conserved motif present in scaffolding and transport proteins across a wide range of organisms. Using hydrogen/deuterium exchange mass spectrometry (HDX MS), a method that has not been widely explored in the context of HEAT repeats, we analysed the structural dynamics of wild-type CNOT1(800–999) and its two double point mutants (E893A/Y900A, E893Q/Y900H) to find the individual contributions of these CNOT1 residues to the molecular recognition of tristetraprolin (TTP). Our results show that the differences in the interactions of CNOT1(800–999) variants with the TTP peptide fragment are due to the absence of the critical residues resulting from point mutations and not due to the perturbation of the protein structure. Nevertheless, the HDX MS was able to detect slight local changes in structural dynamics induced by protein point mutations, which are usually neglected in studies of intermolecular interactions.https://www.mdpi.com/2218-273X/15/3/403CNOT1HEAT repeatTTPhydrogen–deuterium exchangemass spectrometryprotein conformational dynamics |
| spellingShingle | Maja K. Cieplak-Rotowska Michał Dadlez Anna Niedzwiecka Exploring the CNOT1(800–999) HEAT Domain and Its Interactions with Tristetraprolin (TTP) as Revealed by Hydrogen/Deuterium Exchange Mass Spectrometry Biomolecules CNOT1 HEAT repeat TTP hydrogen–deuterium exchange mass spectrometry protein conformational dynamics |
| title | Exploring the CNOT1(800–999) HEAT Domain and Its Interactions with Tristetraprolin (TTP) as Revealed by Hydrogen/Deuterium Exchange Mass Spectrometry |
| title_full | Exploring the CNOT1(800–999) HEAT Domain and Its Interactions with Tristetraprolin (TTP) as Revealed by Hydrogen/Deuterium Exchange Mass Spectrometry |
| title_fullStr | Exploring the CNOT1(800–999) HEAT Domain and Its Interactions with Tristetraprolin (TTP) as Revealed by Hydrogen/Deuterium Exchange Mass Spectrometry |
| title_full_unstemmed | Exploring the CNOT1(800–999) HEAT Domain and Its Interactions with Tristetraprolin (TTP) as Revealed by Hydrogen/Deuterium Exchange Mass Spectrometry |
| title_short | Exploring the CNOT1(800–999) HEAT Domain and Its Interactions with Tristetraprolin (TTP) as Revealed by Hydrogen/Deuterium Exchange Mass Spectrometry |
| title_sort | exploring the cnot1 800 999 heat domain and its interactions with tristetraprolin ttp as revealed by hydrogen deuterium exchange mass spectrometry |
| topic | CNOT1 HEAT repeat TTP hydrogen–deuterium exchange mass spectrometry protein conformational dynamics |
| url | https://www.mdpi.com/2218-273X/15/3/403 |
| work_keys_str_mv | AT majakcieplakrotowska exploringthecnot1800999heatdomainanditsinteractionswithtristetraprolinttpasrevealedbyhydrogendeuteriumexchangemassspectrometry AT michałdadlez exploringthecnot1800999heatdomainanditsinteractionswithtristetraprolinttpasrevealedbyhydrogendeuteriumexchangemassspectrometry AT annaniedzwiecka exploringthecnot1800999heatdomainanditsinteractionswithtristetraprolinttpasrevealedbyhydrogendeuteriumexchangemassspectrometry |