title: Distinct features of cap binding by eIF4E1b proteins.
abstract: eIF4E1b, closely related to the canonical translation initiation factor 4E (eIF4E1a), cap-binding protein is highly expressed in mouse, Xenopus and zebrafish oocytes. We have previously characterized eIF4E1b as a component of the CPEB mRNP translation repressor complex along with the eIF4E-binding protein 4E-Transporter, the Xp54/DDX6 RNA helicase and additional RNA-binding proteins. eIF4E1b exhibited only very weak interactions with m(7)GTP-Sepharose and, rather than binding eIF4G, interacted with 4E-T. Here we undertook a detailed examination of both Xenopus and human eIF4E1b interactions with cap analogues using fluorescence titration and homology modeling. The predicted structure of eIF4E1b maintains the α+β fold characteristic of eIF4E proteins and its cap-binding pocket is similarly arranged by critical amino acids: Trp56, Trp102, Glu103, Trp166, Arg112, Arg157 and Lys162 and residues of the C-terminal loop. However, we demonstrate that eIF4E1b is 3-fold less well able to bind the cap than eIF4E1a, both proteins being highly stimulated by methylation at N(7) of guanine. Moreover, eIF4E1b proteins are distinguishable from eIF4E1a by a set of conserved amino acid substitutions, several of which are located near to cap-binding residues. Indeed, eIF4E1b possesses several distinct features, namely, enhancement of cap binding by a benzyl group at N(7) position of guanine, a reduced response to increasing length of the phosphate chain and increased binding to a cap separated by a linker from Sepharose, suggesting differences in the arrangement of the protein's core. In agreement, mutagenesis of the amino acids differentiating eIF4E1b from eIF4E1a reduces cap binding by eIF4E1a 2-fold, demonstrating their role in modulating cap binding.
authors.affiliation: Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Warsaw 02-089, Poland. Electronic address: dkuba@biogeo.uw.edu.pl.
authors.forename: Dorota
authors.initials: D
authors.lastname: Kubacka
authors.affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK. Electronic address: ricardo_n_miguel@yahoo.com.
authors.forename: Ricardo Núñez
authors.initials: RN
authors.lastname: Miguel
authors.affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK. Electronic address: nicola.minshall@cantab.net.
authors.forename: Nicola
authors.initials: N
authors.lastname: Minshall
authors.affiliation: Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Warsaw 02-089, Poland; Centre of New Technologies, University of Warsaw, Warsaw 02-089, Poland. Electronic address: edek@biogeo.uw.edu.pl.
authors.forename: Edward
authors.initials: E
authors.lastname: Darzynkiewicz
authors.affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK. Electronic address: nms@mole.bio.cam.ac.uk.
authors.forename: Nancy
authors.initials: N
authors.lastname: Standart
authors.affiliation: Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Warsaw 02-089, Poland. Electronic address: jzuberek@biogeo.uw.edu.pl.
authors.forename: Joanna
authors.initials: J
authors.lastname: Zuberek
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D039561:Eukaryotic Initiation Factor-4E / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D014982:Xenopus laevis
chemical_list: D002352:Carrier Proteins; D039561:Eukaryotic Initiation Factor-4E; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C068914:guanosine triphosphate-agarose; D006160:Guanosine Triphosphate; D012685:Sepharose
keywords: Xenopus laevis; binding affinity; eIF4E1b; eukaryotic initiation factor; translational repression