Computational protocol: Distinct Features of Cap Binding by eIF4E1b Proteins

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[…] Multiple sequence alignment was performed using CLUSTALW2. Protein sequences were downloaded from the National Center for Biotechnology Information database and eIF4E/4E-BP-Family Member Database : Homo sapiens 1a: NP_001959.1, 1b: NP_001092878.1; Mus musculus 1a: AAH85087.1, 1b: Q3UTA9.1; X. laevis 1a: NP_001089212.1, 1b: GenBank™ BQ398016 ; Xenopus tropicalis 1a: CAJ83126.1, 1b: AAI54955.1 ; Danio rerio 1a: NP_571808.1, 1b: NP_571529.1; Bos taurus 1a: NP_776735.2, 1b: XP_871211.1; Rattus norvegicus 1a: AAH87001.1, 1b: XP_003753002.1; Canis familiaris 1a: XP_544992.2, 1b: XP_546215.2; Gallus gallus 1a: XP_420655.2, 1b: GenBank™ BX931053.2.Comparative models of the structures of cap-free and cap-bound X. laevis eIF4E1a and eIF4E1b were obtained by the program MODELLER , based on the structure of human cap-free eIF4E1a (PDB ID: 2GPQ) measured in solution and the crystal structure of mouse m7GpppG-bound eIF4E1a (the second guanosine is not visible in the structure; PDB ID: 1L8B ).MODELLER generates protein structures by satisfaction of spatial restraints with simultaneous optimisation of CHARMM energies, conjugate gradients and molecular dynamics with simulated annealing. Comparative models were validated with PROCHECK and WHAT_CHECK that study the geometry of the structures and with VERIFY3D that reports amino acid environmental problems. All protein structure figures were generated using PyMOL [...] Human and Xenopus eIF4E proteins were expressed in the host strain Rosetta2(DE3) (Novagen). Culture of transformed bacteria was induced by 0.5 mM isopropyl-1-thio-β-d-galactopyranoside when the OD600 was ~ 1. After 3 h of incubation at 37 °C, cells were harvested and resuspended in lysis buffer [20 mM Hepes/KOH (pH 7.5), 100 mM KCl, 1 mM EDTA (ethylenediaminetetraacetic acid), 2 mM DTT and 10% (v/v) glycerol] and disrupted by sonication. From the centrifuged lysate (30,000g for 30 min), the supernatant was removed and the pellet was washed three times with wash buffer [1 M guanidine hydrochloride, 20 mM Hepes/KOH (pH 7.2), 2 mM DTT and 10% (v/v) glycerol]. The inclusion bodies were dissolved in buffer containing 6 M guanidine hydrochloride, 50 mM Hepes/KOH (pH 7.2), 10% (v/v) glycerol and 2 mM DTT, and cell debris was removed by centrifugation (43,000g for 30 min). The protein (diluted to a concentration lower than 0.1 mg/mL) was refolded by one-step dialysis against 50 mM Hepes/KOH (pH 7.2), 100 mM KCl, 0.5 mM EDTA and 2 mM DTT and was then purified by ion-exchange chromatography on a HiTrap SP HP column (GE Healthcare) . All eIF4E proteins were eluted with a linear gradient 0.1–1 M KCl in 50 mM Hepes/KOH (pH 7.2) and were analyzed at once in the fluorescent assay without freezing. We added 10% glycerol to the Xenopus eIF4E1b fraction immediately after elution. Additionally for Xenopus eIF4E1b, which possesses more positively charged amino acids than eIF4E1a, the dialysis step was preceded by partially removing the nucleic acid from the protein fraction dissolved in 6 M guanidine hydrochloride on silica membrane (Qiagen).Purity of the collected proteins was assessed by SDS-PAGE electrophoresis and their concentration was determined spectrophotometrically using the extinction coefficients calculated from amino acid compositions with the ProtParam tool (ExPASy Server). These are ε280nm = 53,400 M− 1 cm− 1 for human eIF4E1a, ε280nm = 55,460 M− 1 cm− 1 for human eIF4E1b, ε280nm = 49,960 M− 1 cm− 1 for frog eIF4E1a and its mutated forms and ε280nm = 51,450 M− 1 cm− 1 for eIF4E1bΔN27. […]

Pipeline specifications

Software tools Clustal W, MODELLER, PROCHECK, WHAT_CHECK, VERIFY3D, PyMOL, ProtParam
Databases ExPASy
Applications Protein structure analysis, Protein physicochemical analysis
Organisms Danio rerio, Xenopus laevis, Mus musculus, Homo sapiens
Chemicals Guanine, Phosphates, Sepharose