Changes in conformational equilibria regulate the activity of the Dcp2 decapping enzyme Jan Philip Wurma, Iris Holdermanna, Jan H. Overbecka, Philipp H. O. Mayera, and Remco Sprangersa,1,2 aMax Planck Institute for Developmental Biology, 72076 Tuebingen, Germany Edited by Michael F. Summers, Howard Hughes Medical Institute, University of Maryland, Baltimore County, Baltimore, MD, and approved April 27, 2017 (received for review March 18, 2017) Crystal structures of enzymes are indispensable to understanding Results and Discussion their mechanisms on a molecular level. It, however, remains Structure of the Dcp1:Dcp2:Edc1:m7GDP Complex. Here, we com- challenging to determine which structures are adopted in solution, plement the wealth of structural information on Dcp2 and de- especially for dynamic complexes. Here, we study the bilobed termined the crystal structure of the fully activated Dcp1:Dcp2: decapping enzyme Dcp2 that removes the 5′ cap structure from Edc1:m7GDP complex (Fig. 1 B–D and SI Appendix, Table S1). eukaryotic mRNA and thereby efficiently terminates gene expres- In this structure the guanosine base of m7GDP stacks on Trp-43 of sion. The numerous Dcp2 structures can be grouped into six states the RD and forms specific hydrogen bonds to Asp-47. Residues where the domain orientation between the catalytic and regula- 189–192 of the CD contact the other side of m7GDP via van der tory domains significantly differs. Despite this wealth of structural Waals interactions and thereby sandwich m7GDP between + information it is not possible to correlate these states with the both domains (Fig. 1 B–D). Two Mg2 ions are complexed by the catalytic cycle or the activity of the enzyme. Using methyl trans- phosphate groups of m7GDP and three glutamate residues of verse relaxation-optimized NMR spectroscopy, we demonstrate the catalytic Nudix helix. In addition, residues 157–161 of Edc1 that only three of the six domain orientations are present in so- (including the YAG activation motif Tyr-158–Gly-160) are lution, where Dcp2 adopts an open, a closed, or a catalytically sandwiched between the RD and CD of Dcp2. The Tyr-158 side active state. We show how mRNA substrate and the activator chain stacks between Arg-33 of the RD and Lys-132 of the CD. proteins Dcp1 and Edc1 influence the dynamic equilibria between m7GDP and Edc1 thus stabilize the domain orientation of Dcp2 these states and how this modulates catalytic activity. Impor- by bridging the two Dcp2 domains. tantly, the active state of the complex is only stably formed in The observed domain orientation and product recognition the presence of both activators and the mRNA substrate or the that we observe are very similar to those displayed in the struc- m7GDP decapping product, which we rationalize based on a crys- ture of the Dcp1:Dcp2:Edc3:m7GDP complex (Fig. 1A, orien- tal structure of the Dcp1:Dcp2:Edc1:m7GDP complex. Interest- tation 6a) (13). This is unexpected because Edc3 is unrelated to ingly, we find that the activating mechanisms in Dcp2 also Edc1 and binds to the C-terminal region of Dcp2 far away from result in a shift of the substrate specificity from bacterial to the m7GDP binding site and the interface between the Dcp2 RD eukaryotic mRNA. and CD. Edc3 is thus not able to influence the Dcp2 domain orientation. Surprisingly, the domain orientation in our Dcp1: mRNA decapping | Dcp2 | NMR spectroscopy | catalytic activity | protein dynamics Significance he control of mRNA degradation is essential for the regu- The Dcp2 decapping enzyme targets mRNA for degradation ′ Tlation of gene expression and the removal of the 5 pro- and thereby plays a role in the regulation of gene expression. tective cap of eukaryotic mRNA is a major control step during Despite numerous static crystal structures of the enzyme, it mRNA decay (1). The main decapping enzyme Dcp2 (2, 3) is remained unclear how its catalytic activity correlates with the composed of an N-terminal regulatory domain (RD) and a relative domain orientation in Dcp2. Here we used solution- catalytic Nudix (4) domain (CD) that are connected by a flexible state NMR spectroscopic methods and find that the active state linker (5, 6). The CD shows a basal level of mRNA decapping of Dcp2 is only stably formed in the presence of the Dcp1 and activity, which is increased stepwise upon inclusion of the RD Edc1 activator proteins and the mRNA cap. Importantly, our and upon binding of the activator proteins Dcp1 (6, 7) and Edc1 solution data provide a conclusive view of how the Dcp2 (8–10). Dcp1 binds to the RD of Dcp2 and contains a binding structure changes during the catalytic cycle and provide a platform for proline-rich peptide ligands that can recruit other unique example of the importance of integrated structural bi- mRNA decay factors (9), including the intrinsically disordered ology approaches to unravel the mechanism behind complex decapping enhancer Edc1 that possesses a conserved YAG molecular machines. activation motif located N-terminal to the proline-rich Dcp1 – Author contributions: J.P.W., I.H., J.H.O., and R.S. designed research; J.P.W., I.H., J.H.O., binding sequence (8 10). This motif is essential for decapping P.H.O.M., and R.S. performed research; J.P.W., I.H., J.H.O., and R.S. analyzed data; and enhancement but does not contribute to Dcp1 binding (8, 9, 11). J.P.W. and R.S. wrote the paper. Neither Dcp1 nor Edc1 interacts with the CD of Dcp2 and the The authors declare no conflict of interest. molecular basis for their decapping activation is currently not This article is a PNAS Direct Submission. clear (9). A large number of crystal structures have been de- Freely available online through the PNAS open access option. termined for Dcp2 and include the apo enzyme (12), the Dcp1: Data deposition: Crystallography, atomic coordinates, and structure factors of the Dcp1: Dcp2 complex (6, 10, 13), the Dcp1:Dcp2:Edc1 complex (10), Dcp2:Edc1:m7GDP structure have been deposited in the Protein Data Bank, www.pdb.org Dcp1:Dcp2:Edc1 bound to a substrate analog (11) (Fig. 1A), (PDB ID code 5N2V). 1 and Dcp1:Dcp2 bound to the reaction product m7GDP (Fig. Present address: Department of Biophysics I, University of Regensburg, 93053 Regensburg, Germany. 1A) (13). This latter structure also contains the unrelated 2To whom correspondence should be addressed. Email: [email protected] decapping enhancer Edc3 that binds to Dcp2 C-terminal of regensburg.de. the CD (14) and that does not influence the Dcp2 domain This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. orientation. 1073/pnas.1704496114/-/DCSupplemental. 6034–6039 | PNAS | June 6, 2017 | vol. 114 | no. 23 www.pnas.org/cgi/doi/10.1073/pnas.1704496114 Downloaded by guest on September 29, 2021 Dcp1:Dcp2:ATP Dcp1:Dcp2:Edc1: Dcp2 Dcp1:Dcp2 Dcp1:Dcp2 Dcp1:Dcp2:Edc1 Dcp1:Dcp2:m7GDP A cap analog Dcp1 Edc1 Dcp2 RD Dcp2 CD Nudix helix Nucleotides Orientation 1a and 1b Orientation 2 Orientation 3 Orientation 4 Orientation 5 Orientation 6a Leu 178 C BCEdc1 Dcp1 DTrp 43 Glu 146 Dcp1 Edc1 Mg2+ 180° m7 Edc1 Glu 143 Nudix m7GDP Lys 132 helix Nudix Phe 163 N Helix Glu 147 N Asp 47 Glu 192 Pγ Pβ Dcp2 RD Dcp2 CD Ile 156 E 50 b Orientation 1 6 b a Orientation 6b Pro 161 a Gly 160 0 O CH3 Ala 159 N+ 2 NH O O O Tyr 158 3 BASE 2NH N N O O P O P O P O O Leu 157 -50 4 OHOH OH PC2 (17.49%) YAG activation motif OH OH OO H Dcp2 CD Dcp2 RD 5 m7GDP RNA m7GDP Ile 96 Tyr 92 Arg 33 -50 0 50 PC1 (70.73%) Fig. 1. Static structures of the Dcp2 enzyme. (A) Known structures of Dcp2 display significantly different orientations of the Dcp2 RD (yellow) and CD (green). The catalytic Nudix helix in the CD is indicated in red, Dcp1 in gray, Edc1 in pink, and nucleotides in blue [orientation 1a, PDB ID code 2QKM (6); orientation 1b, 5KQ4 (11); orientation 2, 2A6T (12); orientation 3, 5LON (13); orientation 4, 2QKM (6); orientation 5, 5J3T (10); and orientation 6a, 5LOP (13)]. Note that the protein Edc3 that is present in the structure in orientation 6a is not shown for clarity. (B) Structure of the fully activated Dcp1:Dcp2:Edc1:m7GDP decapping complex (SI Appendix, Table S1). The YAG activation motif in Edc1 docks onto the C-terminal domain of Dcp2 and thereby locks Dcp2 such that the Nudix helix is in close proximity to the substrate binding site. The structure of the 5′ cap is shown and the bond that is hydrolyzed by Dcp2 is indicated. (C) Enlargement of the boxed region in B. Tyr-158 in the Edc1 YAG activation motif stacks between Arg-33 and Lys-132 in Dcp2. (D) The methylated base of the mRNA cap structure stacks onto Trp-43 in the Dcp2 RD and forms hydrogen bonds with Asp-47. Catalytically important Mg2+ ions are coordinated by Glu- 143, 146, and 147 in the Dcp2 Nudix helix. (E) Based on a principle component analysis the known Dcp2 complexes can be separated into six groups. Dcp2:Edc1:m7GDP structure differs significantly from the ori- undergo linearly progressing CSPs upon inclusion of the Dcp2 entation observed in the Dcp1:Dcp2:Edc1 complex in the pres- RD and addition of Dcp1 (Fig. 2A). This suggests a stepwise ence of substrate analog (11) (Fig. 1A, orientation 1b), although increase in the interactions between the Dcp2 RD and CD and a the base of the substrate analog makes similar contacts with the transition from a free (or open) toward a bound (or closed) state.