Crystallographic and Mass Spectrometric Characterisation of eIF4E with N7-alkylated Cap Derivatives
ABSTRACT Structural complexes of the eukaryotic translation initiation factor 4E (eIF4E) with a series of N(7)-alkylated guanosine derivative mRNA cap analogue structures have been characterised. Mass spectrometry was used to determine apparent gas-phase equilibrium dissociation constants (K(d)) values of 0.15 microM, 13.6 microM, and 55.7 microM for eIF4E with 7-methyl-GTP (m(7)GTP), GTP, and GMP, respectively. For tight and specific binding to the eIF4E mononucleotide binding site, there seems to be a clear requirement for guanosine derivatives to possess both the delocalised positive charge of the N(7)-methylated guanine system and at least one phosphate group. We show that the N(7)-benzylated monophosphates 7-benzyl-GMP (Bn(7)GMP) and 7-(p-fluorobenzyl)-GMP (FBn(7)GMP) bind eIF4E substantially more tightly than non-N(7)-alkylated guanosine derivatives (K(d) values of 7.0 microM and 2.0 microM, respectively). The eIF4E complex crystal structures with Bn(7)GMP and FBn(7)GMP show that additional favourable contacts of the benzyl groups with eIF4E contribute binding energy that compensates for loss of the beta and gamma-phosphates. The N(7)-benzyl groups pack into a hydrophobic pocket behind the two tryptophan side-chains that are involved in the cation-pi stacking interaction between the cap and the eIF4E mononucleotide binding site. This pocket is formed by an induced fit in which one of the tryptophan residues involved in cap binding flips through 180 degrees relative to structures with N(7)-methylated cap derivatives. This and other observations made here will be useful in the design of new families of eIF4E inhibitors, which may have potential therapeutic applications in cancer.
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- "Previously it was proposed that dephosphorylation of the eIF4E binding protein 4E-BP1 contributes to the reduction in host protein translation during VSV infection (Connor and Lyles, 2002). The data presented here, together with that published by others (Black et al., 1994; Connor and Lyles, 2002, 2005; Connor et al., 2006; Whitlow et al., 2006, 2008), suggests that M protein may support VSV translation at later times post-infection by compensating for the alteration of the eIF4F cap-binding complex The hypothesis that M may be able to substitute for or enhance eIF4E activity is intriguing since the two proteins are similar in size, both have an unstructured N-terminus, and have similar crystal structures (Brown et al., 2007; Gaudier et al., 2002). Whether the assembly and translational phenotypes of the LXD mutants described here can be separated genetically will require additional investigation. "
ABSTRACT: To study the contribution of the protease-sensitive loop of the VSV M protein in virus assembly we recovered recombinant VSV (rVSV) with mutations in this region and examined virus replication. Mutations in the highly conserved LXD motif (aa 123-125) resulted in reduced virion budding, reduced virus titers and enhanced M protein exchange with M-ribonucleocapsid complexes (M-RNPs), suggesting that the mutant M proteins were less tightly associated with RNP skeletons. In addition, viral protein synthesis began to decrease at 4h post-infection (hpi) and was reduced by ~80% at 8 hpi for the mutant rVSV-D125A. The reduced protein synthesis was not due to decreased VSV replication or transcription; however, translation of a reporter gene with an EMCV IRES was not reduced, suggesting that cap-dependent, but not cap-independent translation initiation was affected in rVSV-D125A infected cells. These results indicate that the LXD motif is involved in both virus assembly and VSV protein translation.Virology 07/2011; 416(1-2):16-25. DOI:10.1016/j.virol.2011.04.013 · 3.28 Impact Factor
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- "-GMP was À12.12. With regard to the substitution effect, fluoro-and chloro-substituted GMPs demonstrated predicted binding modes similar to those observed in the crystal structure (2V8X.pdb), incorporating the key p-cation interaction as well as hydrogen bonding to the phosphate group (Fig. 1). "
ABSTRACT: Aberrant regulation of cap-dependent translation has been frequently observed in the development of cancer. Association of the cap-binding protein eIF4E with N(7)-methylated guanosine capped mRNA is the rate limiting step governing translation initiation; and therefore represents an attractive process for cancer drug discovery. Previously, replacement of the 7-Me group of the Me(7)-guanosine monophosphate with a benzyl group has been found to increase binding affinity to eIF4E. Recent X-ray crystallographic studies have revealed that the cap-binding pocket undergoes a unique structural change in order to accommodate the benzyl group. To explore the structure-activity relationships governing the affinity of N(7)-benzylated guanosine monophosphate (Bn(7)-GMP) for eIF4E, we virtually screened a library of 80 Bn(7)-GMP analogs utilizing CombiGlide as implemented in Schrodinger. A subset library of substituted Bn(7)-GMP analogs was synthesized and their dissociation constants (K(d)) were determined. Due to the poor correlation between docking/scoring results and experimental binding affinities, three-dimensional quantitative structure-activity relationship (3D-QSAR) calculations were performed. Two highly predictive and self-consistent CoMFA (comparative molecular field analysis) and CoMSIA (comparative molecular similarity indices analysis) models were derived and optimized. These models may be useful for the future design of eIF4E cap-binding antagonists.European Journal of Medicinal Chemistry 04/2010; 45(4):1304-13. DOI:10.1016/j.ejmech.2009.11.054 · 3.43 Impact Factor
Article: ["Hand in cap"].Soins. Chirurgie 11/2003;