Structure of the human Ccr4-Not nuclease module using X-ray crystallography and electron paramagnetic resonance spectroscopy distance measurements

Zhang, Qionglin; Pavanello, Lorenzo; Potapov, Alexey; Bartlam, Mark; Winkler, Gerlof Sebastiaan

Abstract

Regulated degradation of mature, cytoplasmic mRNA is a key step in eukaryotic gene regulation. This process is typically initiated by the recruitment of deadenylase enzymes by cis-acting elements in the 3' untranslated region resulting in the shortening and removal of the 3′ poly(A) tail of the target mRNA. The Ccr4-Not complex, a major eukaryotic deadenylase, contains two exoribonuclease subunits with selectivity toward poly(A): Caf1 and Ccr4. The Caf1 deadenylase subunit binds the MIF4G domain of the large subunit CNOT1 (Not1) that is the scaffold of the complex. The Ccr4 nuclease is connected to the complex via its leucine-rich repeat (LRR) domain, which binds Caf1, whereas the catalytic activity of Ccr4 is provided by its EEP domain. While the relative positions of the MIF4G domain of CNOT1, the Caf1 subunit, and the LRR domain of Ccr4 are clearly defined in current models, the position of the EEP nuclease domain of Ccr4 is ambiguous. Here, we use X-ray crystallography, the AlphaFold resource of predicted protein structures, and pulse electron paramagnetic resonance spectroscopy to determine and validate the position of the EEP nuclease domain of Ccr4 resulting in an improved model of the human Ccr4-Not nuclease module.

Citation

Zhang, Q., Pavanello, L., Potapov, A., Bartlam, M., & Winkler, G. S. (2022). Structure of the human Ccr4-Not nuclease module using X-ray crystallography and electron paramagnetic resonance spectroscopy distance measurements. Protein Science, 31(3), 758-764. https://doi.org/10.1002/pro.4262

• Journal Article Type: Article
• Acceptance Date: Dec 14, 2021
• Online Publication Date: Dec 19, 2021
• Publication Date: 2022-03
• Deposit Date: Dec 20, 2021
• Publicly Available Date: Dec 20, 2022
• Journal: Protein Science
• Print ISSN: 0961-8368
• Electronic ISSN: 1469-896X
• Publisher: Wiley
• Peer Reviewed: Peer Reviewed
• Volume: 31
• Issue: 3
• Pages: 758-764
• DOI: https://doi.org/10.1002/pro.4262
• Keywords: Molecular Biology; Biochemistry
• Public URL: https://nottingham-repository.worktribe.com/output/7052564
• Publisher URL: https://onlinelibrary.wiley.com/doi/abs/10.1002/pro.4262
• Additional Information: This is the peer reviewed version of the following article: Zhang, Q., Pavanello, L., Potapov, A., Bartlam, M. and Winkler, G.S. (2021), Structure of the human Ccr4-Not nuclease module using X-ray crystallography and electron paramagnetic resonance spectroscopy distance measurements. Protein Science., which has been published in final form at https://doi.org/10.1002/pro.4262. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited.