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

Abstract

Crystal structures of enzymes are indispensable to understanding their mechanisms on a molecular level. It, however, remains challenging to determine which structures are adopted in solution, especially for dynamic complexes. Here, we study the bilobed decapping enzyme Dcp2 that removes the 5′ cap structure from eukaryotic mRNA and thereby efficiently terminates gene expression. The numerous Dcp2 structures can be grouped into six states where the domain orientation between the catalytic and regulatory domains significantly differs. Despite this wealth of structural information it is not possible to correlate these states with the catalytic cycle or the activity of the enzyme. Using methyl transverse relaxation-optimized NMR spectroscopy, we demonstrate that only three of the six domain orientations are present in solution, where Dcp2 adopts an open, a closed, or a catalytically active state. We show how mRNA substrate and the activator proteins Dcp1 and Edc1 influence the dynamic equilibria between these states and how this modulates catalytic activity. Importantly, the active state of the complex is only stably formed in the presence of both activators and the mRNA substrate or the m7GDP decapping product, which we rationalize based on a crystal structure of the Dcp1:Dcp2:Edc1:m7GDP complex. Interestingly, we find that the activating mechanisms in Dcp2 also result in a shift of the substrate specificity from bacterial to eukaryotic mRNA. mRNA decapping | Dcp2 | NMR spectroscopy | catalytic activity | protein dynamics

Control of mRNA decapping by autoinhibition

“…To corroborate that mutation of Y220 quenched the collective dynamics, we used CPMG spectroscopy on 13 C-methyl ILVMA side-chain labeled Dcp2. Consistent with previous experiments, significant dephasing of transverse magnetization from collective ms-µs motions in wild-type Dcp2 could be attenuated with increasing CPMG pulse rate (Fig 4B, black filled circles) (18,19). In contrast to the wild-type, these dynamics are not present for the Y220G mutant (Fig 4B, blue circles).…”

“…Decapping by Dcp1:Dcp2 requires formation of a composite active site shaped by residues lining the NRD and the CD of Dcp2. The core complex is dynamic in solution, sampling an open form where residues that line the composite active site are far apart, or a closed form where residues on the NRD that engage cap to promote catalysis are occluded (18). Several observations suggest the closed, cap-occluded form of Dcp1:Dcp2 is representative of the autoinhibited form containing the C-terminal extension (Fig 8A).…”

“…We and others have suggested that the ATP-bound, closed Dcp1:Dcp2 structure (Song et al 2008, PDB 2QKM) could resemble an inactive conformation of the enzyme since the substrate binding site is occluded (20,39). Recently, it was shown by NMR that this closed form of Dcp1:Dcp2 is significantly populated in solution (18). We hypothesized the autoinhibited form of Dcp1:Dcp2 might cor-IM1 IM2…”

“…To test this possibility, we used dynamic NMR spectroscopy. Previously, it had been noted that Dcp2 experiences global motions on the millisecond to microsecond timescale (ms-μs dynamics) where the NRD and the CD sample open and closed, cap-occluded forms, with Dcp1 enhancing the population of the latter state (18,19). W43 was identified as a "gatekeeper" required for these dynamics (19).…”

A metal-dependent switch moderates activity of the hexameric M17 aminopeptidases

“…Our appreciation of protein flexibility and dynamics has grown remarkably from the early static lock-and-key model, to our current understanding that proteins are dynamic entities capable of extreme flexibility. For enzymes, flexibility influences substrate binding (50,51) and reaction mechanism (52,53), allowing precise control of reactions that if left unregulated, could be harmful to the cell.…”