Crystal structure of an eIF4G‐like protein from <i>Danio rerio</i>

Abstract

The gene LOC791917 Danio rerio (zebrafish) encodes a protein annotated in the UniProt knowledgebase1 as the “middle domain of eukaryotic initiation factor 4G domain containing protein b” (MIF4Gdb). Its molecular weight is 25.8 kDa, and it comprises 222 amino acid residues. BLAST searches revealed homologues of D. rerio MIF4Gdb in many eukaryotes including humans.2 The homologues and MIF4Gdb were identified as members of the Pfam family, MIF4G (PF02854), which is named after the middle domain of eukaryotic initiation factor 4G (eIF4G).3-5 eIF4G is a component of eukaryotic translational initiation complex, and contains binding sites for other initiation factors, suggesting its critical role in translational initiation.6 The MIF4G domain also occurs in several other proteins involved in RNA metabolism, including the Nonsense-mediated mRNA decay 2 protein (NMD2/UPF2), and the nuclear cap-binding protein 80-kDa subunit (CBP80).5 Sequence and structure analysis of the MIF4G domains in many proteins indicate that the domain assumes an all helical fold and has tandem repeated motifs.5, 7 The zebrafish protein described here has homology to domains of other proteins variously referred to as NIC-containing proteins (NMD2, eIF4G, CBP80). The biological function of D. rerio MIF4Gdb has not yet been experimentally characterized, and the annotation is based on amino acid sequence comparison. D. rerio MIF4Gdb did not share more than 25% sequence identity with any protein for which the three-dimensional structure is known and was selected as a target for structure determination by the Center for Eukaryotic Structural Genomics (CESG). Here, we report the crystal structure of D. rerio MIF4Gdb (UniGene code Dr.79360, UniProt code Q5EAQ1, CESG target number GO.79294). The gene coding for MIF4Gdb was selected as a target as part of a group of genes chosen to code for proteins that are as dissimilar as possible to structures previously deposited in the Protein Data Bank, and also as dissimilar as possible to targets that CESG had previously selected. It was assigned a project database identifier of GO.79294. Complete, detailed protocols for the production of this protein can be found in PepcDB.8 Briefly, the gene was cloned into pVP33K, the first production Flexi®Vector9 used on our project, and selenomethionyl protein was purified following the standard CESG pipeline protocol for cloning,10 protein expression,11 protein purification12 and overall information management.13 Initial crystallization screens were conducted at 4°C and 20°C, in Corning 3775 plates, using a local screen called UW-192. Crystal growth was monitored using Bruker Nonius Crystal Farms at 4°C and 20°C, and scored using Crystal Farm Navigator (Nexus Biosystems). A Tecan Genesis RSP 150 robot assembled precipitant solutions for optimization experiments. Diffraction quality crystals were grown in hanging drop batch experiments. Crystals were grown from 10 mg/mL protein solution in buffer (50 mM NaCl, 3 mM NaN3, 0.3 mM tris(2-carboxyethyl)phosphine (TCEP), 5 mM Bis-Tris pH 7.0) mixed with an equal amount of reservoir solution containing 7% (w/v) poly(ethylene glycol) (PEG) 4K, 0.4 M NaCl, 100 mM MES/acetate pH 5.5 at 20°C. The crystals were cryoprotected in 15% (w/v) ethylene glycol, 10% (w/v) PEG 4K, 100 mM MES/acetate pH 5.5 and were flash-frozen in liquid nitrogen. Diffraction data were collected at Southeast Regional Collaborative Access Team 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory at 100 K. The diffraction images were processed with HKL2000.14 The selenium substructure of the crystal was determined by using SHELXD15 and HySS from PHENIX,16, 17 and the selenium positions were used for single wavelength anomalous diffraction phasing in autoSHARP.18 The initial model was built by the automatic tracing procedure of ARP/wARP,19 and the structure was completed using alternating cycles of manual building in COOT20 and refinement in REFMAC5.21 The st