Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5' cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins

J Mol Biol. 2002 Jun 7;319(3):615-35. doi: 10.1016/S0022-2836(02)00328-5.

Abstract

mRNA 5'-cap recognition by the eukaryotic translation initiation factor eIF4E has been exhaustively characterized with the aid of a novel fluorometric, time-synchronized titration method, and X-ray crystallography. The association constant values of recombinant eIF4E for 20 different cap analogues cover six orders of magnitude; with the highest affinity observed for m(7)GTP (approximately 1.1 x 10(8) M(-1)). The affinity of the cap analogues for eIF4E correlates with their ability to inhibit in vitro translation. The association constants yield contributions of non-covalent interactions involving single structural elements of the cap to the free energy of binding, giving a reliable starting point to rational drug design. The free energy of 7-methylguanine stacking and hydrogen bonding (-4.9 kcal/mol) is separate from the energies of phosphate chain interactions (-3.0, -1.9, -0.9 kcal/mol for alpha, beta, gamma phosphates, respectively), supporting two-step mechanism of the binding. The negatively charged phosphate groups of the cap act as a molecular anchor, enabling further formation of the intermolecular contacts within the cap-binding slot. Stabilization of the stacked Trp102/m(7)G/Trp56 configuration is a precondition to form three hydrogen bonds with Glu103 and Trp102. Electrostatically steered eIF4E-cap association is accompanied by additional hydration of the complex by approximately 65 water molecules, and by ionic equilibria shift. Temperature dependence reveals the enthalpy-driven and entropy-opposed character of the m(7)GTP-eIF4E binding, which results from dominant charge-related interactions (DeltaH degrees =-17.8 kcal/mol, DeltaS degrees= -23.6 cal/mol K). For recruitment of synthetic eIF4GI, eIF4GII, and 4E-BP1 peptides to eIF4E, all the association constants were approximately 10(7) M(-1), in decreasing order: eIF4GI>4E-BP1>eIF4GII approximately 4E-BP1(P-Ser65) approximately 4E-BP1(P-Ser65/Thr70). Phosphorylation of 4E-BP1 at Ser65 and Thr70 is insufficient to prevent binding to eIF4E. Enhancement of the eIF4E affinity for cap occurs after binding to eIF4G peptides.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Carrier Proteins / chemistry*
  • Carrier Proteins / metabolism*
  • Cell Cycle Proteins
  • Crystallography, X-Ray
  • Electrolytes / metabolism
  • Eukaryotic Initiation Factor-4E
  • Eukaryotic Initiation Factor-4G
  • Eukaryotic Initiation Factors
  • Hydrogen Bonding
  • Methylation
  • Mice
  • Models, Molecular
  • Molecular Sequence Data
  • Osmolar Concentration
  • Peptide Fragments / chemical synthesis
  • Peptide Fragments / chemistry
  • Peptide Fragments / metabolism
  • Peptide Initiation Factors / chemistry*
  • Peptide Initiation Factors / metabolism*
  • Phosphates / metabolism
  • Phosphoproteins / chemistry*
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Protein Binding
  • Protein Structure, Tertiary
  • RNA Cap Analogs / chemistry
  • RNA Cap Analogs / metabolism
  • RNA Cap-Binding Proteins
  • RNA Caps / chemistry*
  • RNA Caps / metabolism*
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / metabolism
  • Static Electricity
  • Structure-Activity Relationship
  • Thermodynamics

Substances

  • Carrier Proteins
  • Cell Cycle Proteins
  • Eif4ebp1 protein, mouse
  • Electrolytes
  • Eukaryotic Initiation Factor-4E
  • Eukaryotic Initiation Factor-4G
  • Eukaryotic Initiation Factors
  • Peptide Fragments
  • Peptide Initiation Factors
  • Phosphates
  • Phosphoproteins
  • RNA Cap Analogs
  • RNA Cap-Binding Proteins
  • RNA Caps
  • RNA-Binding Proteins
  • 7-methylguanosine triphosphate