1‐Hydroxy‐xanthine derivatives inhibit the human Caf1 nuclease and Caf1‐containing nuclease complexes via Mg²⁺‐dependent binding

Abstract

In eukaryotic cells, cytoplasmic mRNA is characterised by a 3′ poly(A) tail. The shortening and removal of poly(A) tails (deadenylation) by the Ccr4‐Not nuclease complex leads to reduced translational efficiency and RNA degradation. Using recombinant human Caf1 (CNOT7) enzyme as a screening tool, we recently described the discovery and synthesis of a series of substituted 1‐hydroxy‐3,7‐dihydro‐1H‐purine‐2,6‐diones (1‐hydroxy‐xanthines) as inhibitors of the Caf1 catalytic subunit of the Ccr4‐Not complex. Here, we used a chemiluminescence‐based AMP detection assay to show that active 1‐hydroxy‐xanthines inhibit both isolated Caf1 enzyme and human Caf1‐containing complexes that also contain the second nuclease subunit Ccr4 (CNOT6L) to a similar extent, indicating that the active site of the Caf1 nuclease subunit does not undergo substantial conformational change when bound to other Ccr4‐Not subunits. Using differential scanning fluorimetry, we also show that binding of active 1‐hydroxy‐xanthines requires the presence of Mg2+ ions, which are present in the active site of Caf1.

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

“…[24][25][26][27][28] Biochemical studies using the purified complex, or the isolated nuclease subcomplex also showed a differential contribution of the subunits, although evidence for allosteric regulation has also been reported. [29][30][31][32][33]21 A model based on the Xray structures of the yeast Not1(MIF4G)-Caf1-Ccr4 complex 17 and human CNOT6L 34 suggested interactions between the LRR of Ccr4 and the catalytic EEP domain and a distance of 60 Å between the tightly bound divalent metals in the catalytic sites of Caf1 and Ccr4. 17 By contrast, a recently reported structure of human Caf1/ CNOT7-Ccr4/CNOT6 shows the distance to be notably shorter (46 Å).…”