Molecular mechanism for the inhibition of DXO by adenosine 3′,5′-bisphosphate

Highlights: • pAp inhibits the exoribonuclease activity of DXO. • Crystal structure of DXO was obtained in complex with pAp and Mg²⁺ at 1.8 Å resolution. • pAp competitively inhibits DXO nuclease activity by sharing the binding site pocket. The decapping exoribonuclease DXO functions in pre-mRNA capping quality control, and shows multiple biochemical activities such as decapping, deNADding, pyrophosphohydrolase, and 5′-3′ exoribonuclease activities. Previous studies revealed the molecular mechanisms of DXO based on the structures in complexes with a product, substrate mimic, cap analogue, and 3′-NADP⁺. Despite several reports on the substrate-specific reaction mechanism, the inhibitory mechanism of DXO remains elusive. Here, we demonstrate that adenosine 3′, 5′-bisphosphate (pAp), a known inhibitor of the 5′-3′ exoribonuclease Xrn1, inhibits the nuclease activity of DXO based on the results of structural and biochemical experiments. We determined the crystal structure of the DXO–pAp-Mg²⁺ complex at 1.8 Å resolution. In comparison with the DXO–RNA product complex, the position of pAp is well superimposed with the first nucleotide of the product RNA in the vicinity of two magnesium ions. Furthermore, biochemical assays showed that the inhibition by pAp is comparable between Xrn1 and DXO. Collectively, these structural and biochemical studies reveal that pAp inhibits the activities of DXO by occupying the active site to act as a competitive inhibitor.