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. 2006 Jan;13(1):63-70.
doi: 10.1038/nsmb1033. Epub 2005 Dec 11.

Crystal structure and functional analysis of Dcp2p from Schizosaccharomyces pombe

Meipei She  1 , Carolyn J Decker, Nan Chen, Suneeta Tumati, Roy Parker, Haiwei Song
Affiliations

Crystal structure and functional analysis of Dcp2p from Schizosaccharomyces pombe

Meipei She et al. Nat Struct Mol Biol. 2006 Jan.

Abstract

Decapping is a key step in both general and nonsense-mediated 5' --> 3' mRNA-decay pathways. Removal of the cap structure is catalyzed by the Dcp1-Dcp2 complex. The crystal structure of a C-terminally truncated Schizosaccharomyces pombe Dcp2p reveals two distinct domains: an all-helical N-terminal domain and a C-terminal domain that is a classic Nudix fold. The C-terminal domain of both Saccharomyces cerevisiae and S. pombe Dcp2p proteins is sufficient for decapping activity, although the N-terminal domain can affect the efficiency of Dcp2p function. The binding of Dcp2p to Dcp1p is mediated by a conserved surface on its N-terminal domain, and the N-terminal domain is required for Dcp1p to stimulate Dcp2p activity. The flexible nature of the N-terminal domain relative to the C-terminal domain suggests that Dcp1p binding to Dcp2p may regulate Dcp2p activity through conformational changes of the two domains.

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Figures

Figure 1
Figure 1
Crystal structure of spDcp2n and comparison with other Nudix enzymes. (a) Schematic representation of the domain organization of Dcp2p from S. pombe. (b) Ribbon diagram of spDcp2n. Orange, the N-terminal helical domain; light green, the C-terminal Nudix domain; red, the Nudix motif. Secondary structure elements are labeled. (c) Structure of Ap4AP in complex with a phosphate and AMP (PDB entry 1KTG). Sticks, AMP and phosphates; purple, the Nudix motif. (d) Structure of ADPRP in complex with ADP-ribose (PDB entry 1G9Q). Green, subunit A; gray, subunit B; sticks, ADP-ribose; magenta, the Nudix motif. The view of the Nudix domain for subunit A is the same as those for spDcp2n and Ap4AP.
Figure 2
Figure 2
The Nudix motif of spDcp2n is a catalytic center. (a) Superposition of the Nudix motifs of spDcp2n (purple) and Ap4AP (green). Residues involved in catalysis are shown as stick models. (b) In vitro decapping assays of wild-type spDcp2n and mutant spDcp2n proteins in which conserved glutamate residues in the Nudix motif were substituted by alanine. (c) Northern analysis of steady-state levels of full-length MFA2 mRNA with poly(G) inserted in the 3′ UTR and of the decay intermediate in cells expressing S. cerevisiae wild-type Dcp2p (scDCP2), no Dcp2p (scdcp2Δ) or Dcp2 proteins with single–amino acid substitutions for glutamate residues in the Nudix motif.
Figure 3
Figure 3
Surface views of spDcp2n. (a) Surface representation of spDcp2n showing the regions of high to low sequence conservation among the eukaryotic Dcp2 proteins. Besides the highly conserved Nudix motif, a large conserved patch in the N-terminal domain is revealed and corresponding residues are labeled. (b) Back view of the molecular surface of spDcp2n showing the sequence conservation. The molecule is rotated 180° along a vertical axis relative to the view in a.
Figure 4
Figure 4
The N-terminal domain affects the efficiency of Dcp2p in vitro. (a) Decapping assays of wild-type spDcp2n and deletion mutants of spDcp2n in the presence of Mg2+, Mn2+ or both. Numbers below lanes in a, b and d indicate decapping activity relative to wild-type proteins (average of at least two independent measurements). (b) Decapping assay in the presence of Mn2+ using purified wild-type scDcp2p residues 1–300 (scDcp2n) or residues 102–300 (scDcp2ΔN). (c) Silver-stain analysis of aliquots of purified scDcp2n and scDcp2ΔN proteins used in the decapping assays in b. (d) Decapping assay in the presence of Mn2+ using purified wild-type scDcp2n or scDcp2n proteins containing single-residue substitutions in the N terminus. (e) Silver-stain analysis of aliquots of the purified wild-type and mutant scDcp2n proteins used in the decapping assays in d.
Figure 5
Figure 5
The N-terminal domain of Dcp2p is required for Dcp1p to stimulate decapping. (a) Effects of spDcp1p on the decapping activities of wild-type and mutant spDcp2n proteins in the presence of Mg2+. Wild-type or mutant spDcp2n protein (3 pmol) plus spDcp1p at a 1-, 3- or 10-fold molar ratio relative to spDcp2n or its mutants were used in the decapping assay; triangles denote increase in spDcp1p concentration. (b) Quantification of the stimulation effects shown in a. (c) Decapping assay of mutant spDcp2n proteins with single mutations in the conserved patch of the N-terminal domain in the absence or presence of spDcp1p. 3 pmol of both wild-type and mutant spDcp2n proteins and 18 pmol spDcp1p were used. (d) Quantification of the decapping activities shown in c. (e) Decapping assay in the presence of Mg2+ using purified wild-type scDcp2n or scDcp2n proteins containing single–amino acid substitutions in the N terminus in the presence of scDcp1p (+) or BSA (−). (f) Quantification of the decapping activities shown in e.
Figure 6
Figure 6
The N terminus of Dcp2p is absolutely required in vivo for decapping. (a) Northern analysis of steady-state level of full-length MFA2 mRNA containing poly(G) in its 3′ UTR and that of its decay intermediate produced by decapping and 5′-to-3′ exonucleolytic digestion, in cells expressing wild-type S. cerevisiae Dcp2p (scDCP2), no Dcp2p (scdcp2Δ) or Dcp2p lacking the N-terminal 101 residues (scdcp2ΔN). (b) Northern analysis of steady-state levels of full-length MFA2 mRNA and decay intermediate in cells expressing scDCP2, scdcp2Δ or scDcp2p proteins with single–amino acid substitutions in the N terminus. (c) Histogram showing the ratio of full-length mRNA to intermediate relative to the ratio seen in wild-type cells (set to 1).
Figure 7
Figure 7
Dcp1p interacts with the N-terminal domain of Dcp2p. (a) GST pull-down results showing that both GST-spDcp2n and GST-Dcp2NT bind recombinant spDcp1p, whereas the binding of spDcp1p to GST-Dcp2CT is greatly reduced. (b) GST pull-down results showing binding of GST-tagged spDcp1p to tag-free wild-type (WT) and mutant spDcp2n proteins with single residues substituted to alanine in the N-terminal conserved patch. Control lane contains purified GST-spDcp1p alone. In a and b, proteins were resolved by SDS-PAGE and visualized by Coomassie blue staining. (c) Two-hybrid analysis of the interaction between scDcp1 fused to the Gal4 DNA-binding domain and scDcp2n fused to the Gal4 activation domain. Also tested in place of the latter were the Gal4 activation domain plasmid with no scDcp2n insert (control), a scDcp2ΔN fusion protein and mutant scDcp2n fusion proteins with single–amino acid substitutions in the N-terminal conserved patch. +His, growth on minimal selective plates containing histidine; +3AT, growth on minimal selective plates containing no histidine and 100 mM 3AT.
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