Crystallographic and Mass Spectrometric Characterisation of eIF4E with N7-alkylated Cap Derivatives

Structural Biochemistry, The University of Edinburgh, Michael Swann Building, King's Buildings, Edinburgh, EH9 3JR, Scotland, UK.
Journal of Molecular Biology (Impact Factor: 4.33). 10/2007; 372(1):7-15. DOI: 10.1016/j.jmb.2007.06.033
Source: PubMed


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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    • "The ability of IFITs to outcompete individual eIF4E is consistent with its modest affinity to m7GTP, for which Kd values in the majority of reports range from 140 to 260 nM, depending on the methodology and conditions used for the analysis (e.g. 48–50). The exact affinity for the cap of eIF4E as a subunit of eIF4F has not been determined, but it appears not to contribute significantly to the overall affinity of eIF4F for mRNA, because the apparent dissociation constants for binding of eIF4F to capped and uncapped mRNAs differ only slightly (18 ± 7 nM and 23 ± 7 nM for β-globin mRNA, respectively) (51). "
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    ABSTRACT: Ribosomal recruitment of cellular mRNAs depends on binding of eIF4F to the mRNA’s 5′-terminal ‘cap’. The minimal ‘cap0’ consists of N7-methylguanosine linked to the first nucleotide via a 5′-5′ triphosphate (ppp) bridge. Cap0 is further modified by 2′-O-methylation of the next two riboses, yielding ‘cap1’ (m7GpppNmN) and ‘cap2’ (m7GpppNmNm). However, some viral RNAs lack 2′-O-methylation, whereas others contain only ppp- at their 5′-end. Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed effectors of innate immunity that inhibit viral replication by incompletely understood mechanisms. Here, we investigated the ability of IFIT family members to interact with cap1-, cap0- and 5′ppp- mRNAs and inhibit their translation. IFIT1 and IFIT1B showed very high affinity to cap-proximal regions of cap0-mRNAs (K1/2,app ∼9 to 23 nM). The 2′-O-methylation abrogated IFIT1/mRNA interaction, whereas IFIT1B retained the ability to bind cap1-mRNA, albeit with reduced affinity (K1/2,app ∼450 nM). The 5′-terminal regions of 5′ppp-mRNAs were recognized by IFIT5 (K1/2,app ∼400 nM). The activity of individual IFITs in inhibiting initiation on a specific mRNA was determined by their ability to interact with its 5′-terminal region: IFIT1 and IFIT1B efficiently outcompeted eIF4F and abrogated initiation on cap0-mRNAs, whereas inhibition on cap1- and 5′ppp- mRNAs by IFIT1B and IFIT5 was weaker and required higher protein concentrations.
    Nucleic Acids Research 12/2013; 42(5). DOI:10.1093/nar/gkt1321 · 9.11 Impact Factor
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    • "Crystallographic data on eIF4E indicates that the cap binding slot is essentially divided into a positively charged region which binds the phosphate bridge, and a hydrophobic region where the charged N7-methyl guanosine cap is stacked between two conserved tryptophan residues through cation-π-π interactions [41]. The N7-methyl group is involved in Van der Waal’s interactions only, and has been shown to be substitutable with various alkyl groups without being deleterious to eIF4E binding [42]. It was therefore proposed that the main purpose of the N7-methyl group is to confer a positive charge to the cap. "
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    ABSTRACT: RNA cap binding proteins have evolved to specifically bind to the N7-methyl guanosine cap structure found at the 5' ends of eukaryotic mRNAs. The specificity of RNA capping enzymes towards GTP for the synthesis of this structure is therefore crucial for mRNA metabolism. The fact that ribavirin triphosphate was described as a substrate of a viral RNA capping enzyme, raised the possibility that RNAs capped with nucleotide analogues could be generated in cellulo. Owing to the fact that this prospect potentially has wide pharmacological implications, we decided to investigate whether the active site of the model Parameciumbursaria Chlorella virus-1 RNA capping enzyme was flexible enough to accommodate various purine analogues. Using this approach, we identified several key structural determinants at each step of the RNA capping reaction and generated RNAs harboring various different cap analogues. Moreover, we monitored the binding affinity of these novel capped RNAs to the eIF4E protein and evaluated their translational properties in cellulo. Overall, this study establishes a molecular rationale for the specific selection of GTP over other NTPs by RNA capping enzyme It also demonstrates that RNAs can be enzymatically capped with certain purine nucleotide analogs, and it also describes the impacts of modified RNA caps on specific steps involved in mRNA metabolism. For instance, our results indicate that the N7-methyl group of the classical N7-methyl guanosine cap is not always indispensable for binding to eIF4E and subsequently for translation when compensatory modifications are present on the capped residue. Overall, these findings have important implications for our understanding of the molecular determinants involved in both RNA capping and RNA metabolism.
    PLoS ONE 09/2013; 8(9):e75310. DOI:10.1371/journal.pone.0075310 · 3.23 Impact Factor
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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. "
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    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 · 3.32 Impact Factor
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