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.