Crystal Structures of Human DcpS in Ligand-free and m7GDP-bound forms Suggest a Dynamic Mechanism for Scavenger mRNA Decapping

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Eukaryotic cells utilize DcpS, a scavenger decapping enzyme, to degrade the residual cap structure following 3′-5′ mRNA decay, thereby preventing the premature decapping of the capped long mRNA and misincorporation of methylated nucleotides in nucleic acids. We report the structures of DcpS in ligand-free form and in a complex with m7GDP. apo-DcpS is a symmetric dimer, strikingly different from the asymmetric dimer observed in the structures of DcpS with bound cap analogues. In contrast, and similar to the m7GpppG–DcpS complex, DcpS with bound m7GDP is an asymmetric dimer in which the closed state appears to be the substrate-bound complex, whereas the open state mimics the product-bound complex. Comparisons of these structures revealed conformational changes of both the N-terminal swapped-dimeric domain and the cap-binding pocket upon cap binding. Moreover, Tyr273 in the cap-binding pocket displays remarkable conformational changes upon cap binding. Mutagenesis and biochemical analysis suggest that Tyr273 seems to play an important role in cap binding and product release. Examination of the crystallographic B-factors indicates that the N-terminal domain in apo-DcpS is inherently flexible, and in a dynamic state ready for substrate binding and product release.

Introduction

mRNA decay plays a key role in post-transcriptional regulation of gene expression. In eukaryotes, two major general mRNA decay pathways have been identified.1 Both pathways are initiated with deadenylation of the 3′-poly(A) tail of mRNAs. In the 5′-3′ decay pathways, the 5′ cap structure can be removed by the Dcp1p/Dcp2p complex following deadenylation, thus exposing the 5′ end to 5′-3′ exoribonuclease activities. In the 3′-5′ decay pathways, deadenylation is followed by exosome-dependent 3′-5′ degradation of mRNA body. In addition, two specialized mRNA decay pathways exist to recognize and degrade aberrant mRNAs. In a process referred to as nonsense-mediated mRNA decay (NMD), transcripts with premature translation termination codons are degraded either by deadenylation-independent decapping (5′-3′ NMD), or by accelerated deadenylation and 3′-5′ exonucleolytic digestion by the exosome (3′-5′ NMD).2, 3, 4, 5, 6 Moreover, mRNAs lacking translation termination codons are recognized and degraded rapidly 3′-5′ by the cytoplasmic exosome.7, 8

Decapping in 5′-3′ mRNA decay pathways and removal of the residual cap structure generated by the 3′-5′ mRNA decay pathways require two different types of decapping enzymes. Hydrolysis of the cap structure in the 5′-3′ mRNA decay pathway requires the Dcp1/Dcp2 complex in yeast or Dcp2 alone in mammals.1 Dcp2p, which contains a Nudix motif found in a class of pyrophosphatases,9 catalyzes the release of m7G diphosphate (m7GDP) and monophosphate-terminated mRNA (pRNA) in a metal-dependent manner.10, 11, 12 Interestingly, Dcp2p is an RNA-binding protein that prefers substrates longer than 25 nucleotides.13, 14 The RNA-binding properties of Dcp2 prevent it from degrading the residual cap structure produced by 3′-5′ degradation and restrict its activity to the capped mRNA only.

Degradation of the residual cap structure of 3′-to-5′ exonucleolytic degradation is carried out by DcpS, the scavenger decapping enzyme identified as hDcpS in human and Dcs1p in yeast.15, 16 DcpS proteins are members of the HIT family of pyrophosphatases and use a histidine triad to carry out catalysis in a metal-independent manner, releasing m7G monophosphate (m7GMP) and diphosphate-terminated mRNA (ppRNA).15, 16, 17 Structural analysis has revealed that HIT proteins exist as a homodimer through formation of a continuous ten-stranded antiparallel β sheet, with each HIT protomer containing an active site and nucleotide-binding pocket that coordinates the pyrophosphate bond with respect to the three histidine residues of the catalytic HIT motif.18, 19, 20 Interestingly, DcpS is unable to decap substrates longer than ten nucleotides,16 thereby preventing DcpS from prematurely decapping mRNAs not targeted for degradation. DcpS may also act in the 5′-3′ mRNA decay pathway by converting m7GDP, a product released by Dcp2p to m7GMP, thereby preventing misincorporation of methylated nucleotides in nucleic acids.21 Further support that DcpS is implicated in the 5′-3′ mRNA decay comes from the observation that DcpS is susceptible to m7GDP competition.16

The crystal structures of human DcpS with substitution of the active-site His277 with asparagine, in complex with either m7GpppG or m7GpppA have been reported recently.22 The structures show that DcpS with bound cap analogue is an asymmetric dimer that simultaneously creates an open non-productive DcpS–cap complex and a closed productive DcpS–cap complex, which differs from the open complex by a 30 Å movement of an N-terminal domain. Combined with mutagenesis and biochemical analysis, an autoregulatory mechanism has been proposed to explain how premature decapping of mRNA can be avoided by blocking the conformational changes that are required to form a closed productive active site capable of cap hydrolysis. These differences suggest that DcpS undergoes significant conformational changes during its cycle of substrate binding, hydrolysis and product release.

To gain a better understanding of the molecular mechanisms by which DcpS functions, we determined the crystal structures of human DcpS in ligand-free form (apo-DcpS) and in complex with m7GDP. apo-DcpS is a symmetric dimer different from the asymmetric dimer observed in the structures of hDcpS with bound cap analogues. The structure of hDcpS with bound m7GDP is similar to that of hDcpS with the bound cap analogue. Structural comparison combined with mutagenesis suggests a dynamic mechanism for substrate binding and catalysis.

Section snippets

Structure determination

The full-length human DcpS consisting of amino acid residues 1–337 was expressed in Escherichia coli and purified to homogeneity. For multiwavelength anomalous dispersion (MAD) phasing, a mutant protein in which two Leu residues (Leu206 and Leu317) were mutated to Met was used to obtain the SeMet-substituted crystals. Since this mutant DcpS retained the same decapping activity as the wild-type protein (data not shown), we designated this mutant DcpS protein as apo-DcpS. Crystals of apo-DcpS

Protein expression and purification

The full-length DcpS (1-337) gene was cloned from human Marathon-Ready™ (Clontech) by PCR amplification and expressed as a glutathione-S-transferase (GST)-fusion protein in E. coli. Cells harboring the GST–DcpS fusion protein were lysed by incubation with lysozyme in a lysis buffer (20 mM Tris–HCl (pH 7.6), 500 mM NaCl, 2 mM DTT, 1 mM EDTA, 0.1 mM PMSF, 2 mM benzamide) followed by sonication. The clarified lysate was loaded onto a glutathione-Sepharose 4B column (Amersham). GST-fusion protein was

Acknowledgements

We thank Dr Nobutaka Shimizu at beamline BL40B2, Spring-8, Japan for assistance and access to synchrotron radiation facilities. This work is supported financially by the Agency for Science, Technology and Research (A* Star) in Singapore (H.S.) and by the Howard Hughes Medical Institute (R.P.).

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