DCP1 Forms Asymmetric Trimers to Assemble Into Active Mrna Decapping Complexes in Metazoa
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DCP1 forms asymmetric trimers to assemble into active mRNA decapping complexes in metazoa Felix Tritschler1, Joerg E. Braun1, Carina Motz, Catia Igreja, Gabrielle Haas, Vincent Truffault, Elisa Izaurralde2, and Oliver Weichenrieder2 Department of Biochemistry, Max Planck Institute for Developmental Biology, Spemannstrasse 35, D-72076 Tu¨bingen, Germany Edited by James E. Dahlberg, University of Wisconsin Medical School, Madison, WI, and approved October 29, 2009 (received for review August 28, 2009) DCP1 stimulates the decapping enzyme DCP2, which removes the DCP1a in mRNA decapping we expressed the domain in E. coli -mRNA 5 cap structure committing mRNAs to degradation. In (DCP1a residues S539 to L582). Using static light scattering mea multicellular eukaryotes, DCP1-DCP2 interaction is stabilized by surements coupled with size exclusion chromatography, we found additional proteins, including EDC4. However, most information unexpectedly that the purified domain forms stable trimers in on DCP2 activation stems from studies in S. cerevisiae, which lacks solution (Table S1). We have termed this domain the DCP1- EDC4. Furthermore, DCP1 orthologs from multicellular eukaryotes trimerization domain (DCP1-TD). Furthermore, although the re- have a C-terminal extension, absent in fungi. Here, we show that in combinant polypeptide contains 51 residues per monomer (i.e., 44 metazoa, a conserved DCP1 C-terminal domain drives DCP1 trim- from DCP1a-TD and seven from the expression vector), NMR erization. Crystal structures of the DCP1-trimerization domain re- spectroscopy yields Ͼ115 peaks in the 15N-HSQC spectrum (Fig. veal an antiparallel assembly comprised of three kinked ␣-helices. S2), suggesting that in solution the trimers are asymmetric (assum- Trimerization is required for DCP1 to be incorporated into active ing a single trimeric assembly). decapping complexes and for efficient mRNA decapping in vivo. We determined the crystal structure of this unusual assembly Our results reveal an unexpected connectivity and complexity of to a resolution of 2.3 Å (Rwork ϭ 20.8, Rfree ϭ 25.2, Table S2). the mRNA decapping network in multicellular eukaryotes, which The structure reveals an unprecedented, antiparallel bundle of likely enhances opportunities for regulating mRNA degradation. three kinked ␣-helices (two up, one down), in which the struc- tural environment for a given side-chain differs for each of the ͉ ͉ ͉ ͉ DCP2 miRNAs P-bodies EDC4 Ge-1 three molecules (Fig. 1 B–F). The central sequence (K544-L571) of each molecule is ␣-helical, with a strong kink at D558 that n eukaryotes, removal of the mRNA 5Ј cap structure is separates helix ␣1 from helix ␣2 with an elbow angle of Ϸ90° Icatalyzed by the decapping enzyme DCP2 (1, 2); to be fully (Fig. 1F). At the kink, residue L554 from helix ␣1 makes van der active and/or stable, DCP2 requires additional proteins (1, 2). Waals contacts with F561 from helix ␣2, while D558 caps the N Yeast DCP2 interacts directly with DCP1 and this interaction is terminus of helix ␣-2 by hydrogen-bonding with the peptide required for decapping in vivo and in vitro (3–7). In humans, the NH-groups of residues S560 and F561 (Fig. 1F). Alignment of DCP2-DCP1 interaction requires additional proteins, which DCP1 sequences from various species shows only these three together assemble into multimeric decapping complexes that residues are invariant (Fig. 2A, asterisks), indicating the elbow is also include the enhancers of decapping 3 and 4 (EDC3 and a conserved structural feature of DCP1 trimerization domains ECD4), and the DEAD-box protein DDX6/RCK (8, 9). from multicellular eukaryotes. DCP2 is highly conserved and most information on DCP2 The three polypeptide chains (termed A, B, and C in Fig. 1 activation stems mainly from studies in S. cerevisiae and S. pombe B–F) superimpose over the central sequence, with a maximal C␣ (3–7). Fungi, however, lack EDC4 as well as many extensions and r.m.s.d. of 1.15 Å (chain A and B, residues 544–571). To additional domains present in decapping activators of metazoan assemble the trimer, chain B interacts with chains A and C in an orthologs (8–10). For example, all eukaryotic DCP1 proteins antiparallel fashion, causing helices ␣1 of chain A and ␣2 of chain contain an N-terminal EVH1 domain (3, 5, 6); however, DCP1 C to interact in parallel. Chains A and B are thus related by a orthologs from metazoa and plants also have a proline-rich pseudotwofold axis close to F561 (Fig. 1D), while chains C and C-terminal extension (9, 10). The sequence of this extension is B are related by a pseudotwofold axis close to I552 (Fig. 1E). not conserved except for a 14-residue short motif (motif I, MI) This arrangement places most hydrophobic side chains into a C. elegans A conserved in metazoa with the exception of (Fig. 1 densely packed core (Figs. 1B and 2A, residues shaded in blue), and Fig. S1) and a C-terminal domain conserved in plants and explaining the stability of the trimer. The resulting DCP1a metazoa (Fig. 1A, referred to as TD). trimerization domain could be characterized as a novel fold if the The DCP1 C-terminal domain is predicted to adopt an chains were connected in cis. ␣-helical conformation. In this work, we show that this domain Using the structural information, we constructed three mu- trimerizes in an asymmetric fashion. We solved the crystal structure of the trimerized domain for both human and D. melanogaster DCP1 and show that the trimer adopts an unprec- Author contributions: E.I. designed research; F.T., J.E.B., C.M., C.I., G.H., V.T., and O.W. edented fold, with no current similarities in the protein database. performed research; F.T., J.E.B., C.M., C.I., G.H., V.T., E.I., and O.W. analyzed data; and F.T., We further show that DCP1 trimerization is required for the J.E.B., E.I., and O.W. wrote the paper. assembly of active decapping complexes and for mRNA decap- The authors declare no conflict of interest. ping in vivo. The conservation of structurally critical residues This article is a PNAS Direct Submission. BIOCHEMISTRY indicates that this domain adopts a similar fold in DCP1 or- Freely available online through the PNAS open access option. thologs of other multicellular eukaryotes. Consequently, within Data deposition: Coordinates of the human DCP1a and the D. melanogaster DCP1 trimer- mRNA decapping complexes in these organisms, the stoichiom- ization domains have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code etry of the protein components is likely more complex than 2WX3 and 2WX4). previously thought. 1F.T. and J.E.B. contributed equally to this work. 2To whom correspondence may be addressed. E-mail: [email protected] Results and Discussion or [email protected]. Crystal Structure of the Human DCP1a Trimerization Domain. To This article contains supporting information online at www.pnas.org/cgi/content/full/ investigate the role of the conserved C-terminal domain of human 0909871106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0909871106 PNAS ͉ December 22, 2009 ͉ vol. 106 ͉ no. 51 ͉ 21591–21596 Downloaded by guest on September 25, 2021 Crystal Structure of the Drosophila melanogaster DCP1 Trimerization Domain. Despite the apparent stability of this unusual asymmet- ric homotrimeric assembly, we could not formally rule out the possibility that this particular protein sequence causes a unique artifact. Therefore, we crystallized the trimerization domain of D. melanogaster DCP1 (residues L328 to D366), which is only 36% identical to the human DCP1a trimerization domain (iden- tity calculated over the central sequence of the TDs: HsDCP1a 544–571, DmDCP1 331–358; Fig. 2A). We found it also trim- erizes with the same topology but in a different crystal packing environment and with two independent copies in the asymmetric unit (Fig. 1C, Fig. S3, and Table S2). Moreover, when D. melanogaster DCP1 is mutated at structural positions equivalent to those in human DCP1a mutants (Mut-1, Mut-2, and Mut-3), trimerization is again disrupted (Table S1). We therefore con- clude that the C terminus of DCP1 contains a trimerization domain, which is conserved in metazoa but absent in fungi. Functional Analysis of the Human and D. melanogaster DCP1 Trimer- ization Domain. Further analysis showed DCP1 also oligomerizes in vivo. In human HEK293 cells, we coexpressed hemagglutinin (HA)-tagged DCP1a with green fluorescent protein (GFP)- tagged wild-type or mutant DCP1a, and then performed coim- munoprecipitations with anti-GFP antibodies. HA-DCP1a co- immunoprecipitated with GFP-DCP1a, but not with the negative control protein GFP-MBP (Fig. 2B, lane 9 vs. 8). As expected, the trimerization domain (GFP-DCP1a-TD) is sufficient for oligomerization (Fig. 2C, lane 6); while for DCP1a mutants 1, 2 and 3 oligomerization is strongly impaired (Fig. 2B, lanes 10–12). Accordingly, deleting the trimerization domain prevents DCP1a oligomerization (Fig. 2B, lane 13). Similarly, we confirmed that the trimerization domain of D. melanogaster DCP1 is also sufficient for oligomerization in vivo (Fig. 2D). Because we were able to disrupt oligomerization in vivo using information from the crystal structure, we infer DCP1 in human and D. melanogaster cells exist as asymmetric trimers. Accord- ingly, the possibility that the observed DCP1a self-association is indirect and results from the incorporation of several monomeric DCP1a copies into larger decapping complexes can be ruled out, as it is inconsistent with the crystal structures and with the findings described below. We next showed human DCP1a trimerization is required for assembly of active decapping complexes. We immunopurified GFP-DCP1a (wild-type and mutants) from HEK293 cells and, using an m7G-capped RNA substrate, tested for decapping activity in vitro. Decapping activity coimmunopurified with Fig.