Conformational dynamics in bilobed enzymes can be used to regulate their activity. One such enzyme, the eukaryotic decapping enzyme Dcp2, controls the half-life of mRNA by cleaving the 5′ cap structure, which exposes a monophosphate that is efficiently degraded by exonucleases. Decapping by Dcp2 is thought to be controlled by an open-to-closed transition involving formation of a composite active site with two domains sandwiching sub-strate, but many details of this process are not understood. Here, using NMR spectroscopy and enzyme kinetics, we show that Trp43 of Schizosaccharomyces pombe Dcp2 is a conserved gatekeeper of this open-to-closed transition. We find that Dcp2 samples multi-ple conformations in solution on the millisecond-microsecond timescale. Mutation of the gatekeeper tryptophan abolishes the dynamic behavior of Dcp2 and attenuates coactivation by a yeast enhancer of decapping (Edc1). Our results determine the dynamics of the open-to-closed transition in Dcp2, suggest a structural path-way for coactivation, predict that Dcp1 directly contacts the cata-lytic domain of Dcp2, and show that coactivation of decapping by Dcp2 is linked to formation of the composite active site. enzyme dynamics ∣ methyl groups ∣ mRNA decay ∣ protein NMR C onformational dynamics in enzymes often comprise the rate-limiting step in the catalytic cycle and thus are prime targets for regulatory cofactors (1–5). Bilobed proteins frequently use an open-to-closed transition to coordinate catalysis on their substrates following cellular cues such as posttranslational mod-ifications or macromolecular interactions (6–8). A recent model proposes that the eukaryotic mRNA decapping enzyme Dcp2 is regulated by such a transition, where a composite active site is formed using conserved surfaces on each of the two N-terminal domains (9). According to this model, stimulating or inhibiting this conformational transition could regulate decapping. How-ever, the structural details of this composite active site and the timescale of interconversion between closed and open states of Dcp2 are currently unknown. Moreover, whether coactivators use the composite active site to effect decapping is unclear. Degradation of eukaryotic mRNA is critical to many biological processes including development (10), stress response (11), clear-ance of the products of pervasive transcription (12), and quality control of gene expression (13). For example, it has been sug-gested that microRNAs (miRNAs) act primarily by destabilizing messages and it is known that Dcp2 is a vital component of miRNA-induced mRNA decay (14, 15). Further, an entire class of unstable transcripts was recently discovered that is sensitive to the exonuclease Xrn1, whose members are therefore likely pro-ducts of decapping (12, 16). Each of the variety of pathways that utilize decapping relies on coactivator proteins that are believed to recruit messages to the decapping machinery and activate it. A model of decapping coactivation is emerging following recent work on the Saccharomyces cerevisiae coactivator Edc1 (17). Edc1 is a yeast-specific protein that is required for carbon source changes and is strongly upregulated during such transi-tions (18–20). It binds directly to Dcp1, which in turn forms a stable complex with the regulatory domain of Dcp2 (17, 21). Dcp1 has an enabled/VASP homology-1 (EVH1) fold and uses a hydrophobic patch to recognize a proline-rich stretch in the C terminus of Edc1, which is also found in other putative coac-tivators (17, 22). Binding of Edc1 raises the catalytic efficiency of the Dcp1∶Dcp2 complex by up to 3,000 times by enhancing both the K M for mRNA and rate of the catalytic step k max (17). Inter-estingly, it appears that Edc1 is modular with the N-terminal re-gion responsible for the K M enhancement and the C-terminal region primarily affecting k max (17). The mechanism of this en-hancement is unknown though it was proposed that Edc1 may stimulate closure and thereby activity of Dcp2. Many proteins are dynamic on the millisecond-microsecond (ms-μs) timescale and these motions can be intimately tied to activity (1). Dcp2 is known to undergo an open-to-closed transi-tion that leads to formation of the composite active site and it was suggested that both the apo and ligand-bound forms of the enzyme sample multiple conformations (9). Open-to-closed tran-sitions in bilobed proteins like Dcp2 can occur on the ms-μs timescale, which can be monitored with site-specific resolution by NMR spectroscopy (23, 24). Motions on the ms-μs timescale lead to dephasing of transverse magnetization which manifests in a rate constant R ex that, together with R 2 for molecular tum-bling, determines the resonance linewidth. Carr–Purcell–Mei-boom–Gill (CPMG) NMR spectroscopy allows contributions to the resonance linewidth from molecular tumbling to be sepa-rated from ms-μs dynamics (25). When coupled with