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PMID: 20628353 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Crystal structure of the human CNOT6L nuclease domain reveals strict poly(A) substrate specificity.

The EMBO journal ·Vol. 29 ·No. 15 ·2010-08-04 ·Pages 2566-76

Wang H, Morita M, Yang X, Suzuki T, Yang W, Wang J, Ito K, Wang Q, Zhao C, Bartlam M, Yamamoto T, Rao Z

Abstract

CCR4, an evolutionarily conserved member of the CCR4-NOT complex, is the main cytoplasmic deadenylase. It contains a C-terminal nuclease domain with homology to the endonuclease-exonuclease-phosphatase (EEP) family of enzymes. We have determined the high-resolution three-dimensional structure of the nuclease domain of CNOT6L, a human homologue of CCR4, by X-ray crystallography using the single-wavelength anomalous dispersion method. This first structure of a deadenylase belonging to the EEP family adopts a complete alpha/beta sandwich fold typical of hydrolases with highly conserved active site residues similar to APE1. The active site of CNOT6L should recognize the RNA substrate through its negatively charged surface. In vitro deadenylase assays confirm the critical active site residues and show that the nuclease domain of CNOT6L exhibits full Mg(2+)-dependent deadenylase activity with strict poly(A) RNA substrate specificity. To understand the structural basis for poly(A) RNA substrate binding, crystal structures of the CNOT6L nuclease domain have also been determined in complex with AMP and poly(A) DNA. The resulting structures suggest a molecular deadenylase mechanism involving a pentacovalent phosphate transition.

MeSH Terms
Biocatalysis Crystallography, X-Ray DNA, Single-Stranded/metabolism Humans Magnesium/metabolism Models, Molecular Nucleic Acid Conformation Poly A/chemistry,metabolism Protein Binding Protein Structure, Tertiary Ribonucleases/chemistry,genetics,metabolism Substrate Specificity
Chemicals
DNA, Single-Stranded Poly A CNOT6L protein, human Ribonucleases Magnesium
Authors & Affiliations
12 authors, click to expand affiliations / ORCID
Wang Hui
Tianjin Key Laboratory of Protein Science, College of Life Sciences, Nankai University, Tianjin, PR China.
Morita Masahiro
Yang Xiuna
Suzuki Toru
Yang Wen
Wang Jiao
Ito Kentaro
Wang Quan
Zhao Cong
Bartlam Mark
Yamamoto Tadashi
Rao Zihe
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Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
1460-2075
DOI
10.1038/emboj.2010.152
Published
2010-08-04
Epub
2010-00-13
Pages
2566-76
Language
English
Region
England
NLM ID
8208664
PMCID
PMC2928688
Subset
IM
External Links
PubMed source DOI: 10.1038/emboj.2010.152 PMC Full Text View journal details
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