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

Hui Wang, Masahiro Morita, Xiuna Yang, Toru Suzuki, Wen Yang, Jiao Wang, Kentaro Ito, Quan Wang, Cong Zhao, Mark Bartlam, Tadashi Yamamoto, Zihe Rao

Research output: Contribution to journalArticlepeer-review

77 Scopus citations

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 α/β 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 Mg2+-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.

Original languageEnglish (US)
Pages (from-to)2566-2576
Number of pages11
JournalEMBO Journal
Volume29
Issue number15
DOIs
StatePublished - Aug 4 2010
Externally publishedYes

Keywords

  • CCR4
  • crystal structure
  • deadenylase
  • mRNA degradation
  • poly(A) substrate

ASJC Scopus subject areas

  • General Immunology and Microbiology
  • General Biochemistry, Genetics and Molecular Biology
  • Molecular Biology
  • General Neuroscience

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