Structure of the active form of Dcp1–Dcp2 decapping enzyme bound to m7GDP and its Edc3 activator

Abstract

Elimination of the 5' cap of eukaryotic mRNAs, known as decapping, is considered to be a crucial, irreversible and highly regulated step required for the rapid degradation of mRNA by Xrn1, the major cytoplasmic 5'-3' exonuclease. Decapping is accomplished by the recruitment of a protein complex formed by the Dcp2 catalytic subunit and its Dcp1 cofactor. However, this complex has a low intrinsic enzymatic activity and requires several accessory proteins such as the Lsm1-7 complex, Pat1, Edc1-Edc2 and/or Edc3 to be fully active. Here we present the crystal structure of the active form of the yeast Kluyveromyces lactis Dcp1-Dcp2 enzyme bound to its product (mGDP) and its potent activator Edc3. This structure of the Dcp1-Dcp2 complex bound to a cap analog further explains previously published data on substrate binding and provides hints as to the mechanism of Edc3-mediated Dcp2 activation.

Changes in conformational equilibria regulate the activity of the Dcp2 decapping enzyme

“…1A, orientation 6a) (13). This is unexpected because Edc3 is unrelated to Edc1 and binds to the C-terminal region of Dcp2 far away from the m7GDP binding site and the interface between the Dcp2 RD and CD.…”

“…Proteins were subsequently purified through NiNTA, cation exchange, and size-exclusion chromatography. For NMR measurements Dcp2 was 1 H 3 - 13 C-labeled at the Ile, Met, Val, Ala, and Leu methyl groups in a fully deuterated background (30). RNA was produced by standard in vitro transcription using T7 polymerase and capped with vaccinia capping enzyme (31).…”

“…In summary, our data demonstrate that the Dcp1:Dcp2 complex adopts the closed orientation 1a and that this structure transiently opens. Interestingly, this orientation is catalytically impaired because the catalytic Nudix helix is remote from the cap binding site (6,11,13) and the RNA binding path on the CD is partially blocked (discussed below). Edc1 Has No Influence on the Dcp1:Dcp2 Structure.…”

“…1A), and Dcp1:Dcp2 bound to the reaction product m7GDP (Fig. 1A) (13). This latter structure also contains the unrelated decapping enhancer Edc3 that binds to Dcp2 C-terminal of the CD (14) and that does not influence the Dcp2 domain orientation.…”

“…Neither Dcp1 nor Edc1 interacts with the CD of Dcp2 and the molecular basis for their decapping activation is currently not clear (9). A large number of crystal structures have been determined for Dcp2 and include the apo enzyme (12), the Dcp1: Dcp2 complex (6,10,13), the Dcp1:Dcp2:Edc1 complex (10), Dcp1:Dcp2:Edc1 bound to a substrate analog (11) (Fig. 1A), and Dcp1:Dcp2 bound to the reaction product m7GDP (Fig.…”