Stopped-flow kinetic analysis of eIF4E and phosphorylated eIF4E binding to cap analogs and capped oligoribonucleotides: evidence for a one-step binding mechanism

Recruitment of eukaryotic mRNA to the 48 S initiation complex is rate-limiting for protein synthesis under normal conditions. Binding of the 5' -terminal cap structure of mRNA to eIF4E is a critical event during this process. Mammalian eIF4E is phosphorylated at Ser-209 by Mnk1 and Mnk2 kinases. We investigated the interaction of both eIF4E and phosphorylated eIF4E (eIF4E(P)) with cap analogs and capped oligoribonucleotides by stopped-flow kinetics. For m(7)GpppG, the rate constant of association, k(on), was dependent on ionic strength, decreasing progressively up to 350 mm KCl, but the rate constant of dissociation, k(off), was independent of ionic strength. Phosphorylation of eIF4E decreased k(on) by 2.1-2.3-fold at 50-100 mm KCl but had progressively less effect at higher ionic strengths, being negligible at 350 mm. Contrary to published evidence, eIF4E phosphorylation had no effect on k(off). Several observations supported a simple one-step binding mechanism, in contrast to published reports of a two-step mechanism. The kinetic function that best fit the data changed from single- to double-exponential as the eIF4E concentration was increased. However, measuring k(off) for dissociation of a pre-formed eIF4E.m(7)GpppG complex suggested that the double-exponential kinetics were caused by dissociation of eIF4E dimers, not a two-step mechanism. Addition of a 12-nucleotide chain to the cap structure increased affinity at high ionic strength for both eIF4E (24-fold) and eIF4E(P) (7-fold), primarily due to a decrease in k(off). This suggests that additional stabilizing interactions between capped oligoribonucleotides and eIF4E, which do not occur with cap analogs alone, act to slow dissociation.     

Stopped-flow fluorescence studies of HMG-domain protein binding to cisplatin-modified DNA

High-mobility group (HMG) domain proteins bind specifically to the major DNA adducts formed by the anticancer drug cisplatin and can modulate the biological response to this inorganic compound. Stopped-flow fluorescence studies were performed to investigate the kinetics of formation and dissociation of complexes between HMG-domain proteins and a series of 16-mer oligonucleotide probes containing both a 1,2-intrastrand d(GpG) cisplatin cross-link and a fluorescein-modified deoxyuridine residue. Rate constants, activation parameters, and dissociation constants were determined for complexes formed by HMG1 domain A and the platinated DNA probes. The sequence context of the cisplatin adduct modulates the value of the associative rate constant for HMG1 domain A by a factor of 2-4, contributing significantly to differences in binding affinity. The rates of association or dissociation of the protein-DNA complex were similar for a 71 bp platinated DNA analogue. Additional kinetic studies performed with HMG1 domain B, an F37A domain A mutant, and the full-length HMG1 protein highlight differences in the binding properties of the HMG domains. The stopped-flow studies demonstrate the utility of the fluorescein-dU probe in studying protein-DNA complexes. The kinetic data will assist in determining what role these proteins might play in the cisplatin mechanism of action.     

Stopped-flow fluorescence studies on binding kinetics of neurotoxins with acetylcholine receptor

Acetylcholine receptor from Narke japonica electroplax exhibits a fluorescence change upon binding of snake neurotoxins. This fluorescence change primarily arises from the conformational change of the acetylcholine receptor and reflects the binding process of the toxin with the receptor. The time dependence of the fluorescence change has been monitored for 28 short neurotoxins and 8 long neurotoxins by using a stopped-flow technique. The steady-state fluorescence change is of the same order of magnitude for the short neurotoxins but varies among the long neurotoxins. Nha 10, a short neurotoxin with weak neurotoxicity, causes no fluorescence change in the receptor but can still bind to the receptor with sufficiently high affinity. The substitution of the conserved residue Asp-31 to Gly-31 in Nha is probably responsible for the reduced neurotoxicity. The rate constants for the binding of the neurotoxins to the receptor have been obtained by analyzing the transient fluorescence change. The rate constants show surprisingly a wide range of distribution: (1.0-20.5) X 10(6) M-1 s-1 for short neurotoxins and (0.26-1.9) X 10(6) M-1 s-1 for long neurotoxins. Examination of the relationship between the rate constants of fluorescence change of the short neurotoxins and their amino acid sequences, thermal stability, hydrogen-deuterium exchange behavior, overall net charge, etc. reveals the following. Positive charges on the side chains of residues 27 and 30 and overall net charge of the neurotoxin govern the magnitude of the binding rate of the neurotoxin with the receptor.     

Synthesis and evaluation of potential inhibitors of eIF4E cap binding to 7-methyl GTP

Cap-dependent translation is initiated by the binding of eIF4E to capped mRNA (m(7)GpppN). We have prepared a small library of 7-methyl guanosine nucleoside and nucleotide analogs and evaluated their ability to inhibit eIF4E binding to 7-methyl GTP with a competitive eIF4E binding immunoassay. 5'-H-Phosphonate derivatives in which the 2'- and 3'-riboside hydroxyls were tethered together by an isopropylidene group were shown to be a new class of inhibitors of eIF4E binding to capped mRNA.     

A microtiter plate assay for assessing the interaction of eukaryotic initiation factor eIF4E with eIF4G and eIF4E binding protein-1

Modulation of interactions among proteins is an important mechanism for regulating both the subcellular location and the function of proteins. An example of the importance of protein-protein interaction is the reversible association of eukaryotic initiation factor eIF4E with the eIF4E binding proteins 4E-BP1 and eIF4G. When bound to 4E-BP1, eIF4E cannot bind to eIF4G to form the active eIF4F complex, an event that is required for the binding of mRNA to the ribosome. Thus, association of eIF4E with 4E-BP1 represses mRNA translation by preventing the binding of mRNA to the ribosome. Previous studies have measured the amount of 4E-BP1 or eIF4G bound to eIF4E by either affinity chromatography or immunoprecipitation of eIF4E followed by Western blot analysis for quantitation of 4E-BP1 and eIF4G. Both of these techniques have significant limitations. In the present study, we describe a microtiter plate-based assay for quantitation of the amount of 4E-BP1 and eIF4G bound to eIF4E that obviates many of the limitations of the earlier approaches. It also has the advantage that absolute amounts of the individual proteins can be easily estimated. The approach should be applicable to the study of a wide variety of protein-protein interactions.     

Vesicular stomatitis virus infection alters the eIF4F translation initiation complex and causes dephosphorylation of the eIF4E binding protein 4E-BP1

Vesicular stomatitis virus (VSV) modulates protein synthesis in infected cells in a way that allows the translation of its own 5'-capped mRNA but inhibits the translation of host mRNA. Previous data have shown that inactivation of eIF2alpha is important for VSV-induced inhibition of host protein synthesis. We tested whether there is a role for eIF4F in this inhibition. The multisubunit eIF4F complex is involved in the regulation of protein synthesis via phosphorylation of cap-binding protein eIF4E, a subunit of eIF4F. Translation of host mRNA is significantly reduced under conditions in which eIF4E is dephosphorylated. To determine whether VSV infection alters the eIF4F complex, we analyzed eIF4E phosphorylation and the association of eIF4E with other translation initiation factors, such as eIF4G and the translation inhibitor 4E-BP1. VSV infection of HeLa cells resulted in the dephosphorylation of eIF4E at serine 209 between 3 and 6 h postinfection. This time course corresponded well to that of the inhibition of host protein synthesis induced by VSV infection. Cells infected with a VSV mutant that is delayed in the ability to inhibit host protein synthesis were also delayed in dephosphorylation of eIF4E. In addition to decreasing eIF4E phosphorylation, VSV infection also resulted in the dephosphorylation and activation of eIF4E-binding protein 4E-BP1 between 3 and 6 h postinfection. Analysis of cap-binding complexes showed that VSV infection reduced the association of eIF4E with the eIF4G scaffolding subunit at the same time as its association with 4E-BP1 increased and that these time courses correlated with the dephosphorylation of eIF4E. These changes in the eIF4F complex occurred over the same time period as the onset of viral protein synthesis, suggesting that activation of 4E-BP1 does not inhibit translation of viral mRNAs. In support of this idea, VSV protein synthesis was not affected by the presence of rapamycin, a drug that blocks 4E-BP1 phosphorylation. These data show that VSV infection results in modifications of the eIF4F complex that are correlated with the inhibition of host protein synthesis and that translation of VSV mRNAs occurs despite lowered concentrations of the active cap-binding eIF4F complex. This is the first noted modification of both eIF4E and 4E-BP1 phosphorylation levels among viruses that produce capped mRNA for protein translation.     

Brownian dynamics study of the influences of electrostatic interaction and diffusion on protein-protein association kinetics.

A unified model is presented for protein-protein association processes that are under the influences of electrostatic interaction and diffusion (e.g., protein oligomerization, enzyme catalysis, electron and energy transfer). The proteins are modeled as spheres that bear point charges and undergo translational and rotational Brownian motion. Before association can occur the two spheres have to be aligned properly to form a reaction complex via diffusion. The reaction complex can either go on to form the product or it can dissociate into the separate reactants through diffusion. The electrostatic interaction, like diffusion, influences every step except the one that brings the reaction complex into the product. The interaction potential is obtained by extending the Kirkwood-Tanford protein model (Tanford, C., and J. G. Kirkwood. 1957. J. Am. Chem. Soc. 79:5333-5339) to two charge-embedded spheres and solving the consequent equations under a particular basis set. The time-dependent association rate coefficient is then obtained through Brownian dynamics simulations according an algorithm developed earlier (Zhou, H.-X. 1990. J. Phys. Chem. 94:8794-8800). This method is applied to a model system of the cytochrome c and cytochrome c peroxidase association process and the results confirm the experimental dependence of the association rate constant on the solution ionic strength. An important conclusion drawn from this study is that when the product is formed by very specific alignment of the reactants, as is often the case, the effect of the interaction potential is simply to scale the association rate constant by a Boltzmann factor. This explains why mutations in the interface of the reaction complex have strong influences on the association rate constant whereas those away from the interface have minimal effects. It comes about because the former mutations change the interaction potential of the reaction complex significantly and the latter ones do not.     

Stopped-flow kinetic studies of actinomycin binding to DNAs.

Stopped-flow kinetic studies of the association of actinomycins with narural and synthetic DNA duplexes are presented. The actinomycins examined were D (C1), D lactam (in which the pentapeptide rings are closed by lactam instead of lactone linkages), X2, XObeta, and actinomine. The DNAs used included claf-thymus DNA, PM2, DNA, and two synthetic d(A-T)-lide copolymers containing 2,6-diaminopurine (DAP) in place of adenine residues, poly[d(DAP-T)]-poly[d(DAP-T)] and poly[d(DAP-A-T]-poly[d(DAP-A-T)]. Apparent equilibrium constants indicate that the DAP-containing polynucleotides bind actinomycin strongly. Comples formation of actinomycins D, D lactam, X2 and XObeta with these DNAs can be deconvoluted into five rate processes. These steps do not necessarily proceed to completion. The rates of two of these steps display a firstorder dependence on DNA concentration. The large negative entropies of activation of these steps suggest a high degree of restriction to freedom of motion on the respective transition states. The rates of the remaining three steps are independent of DNA concentration. Kinetic parameters of actinimycin binding to DNAs are presented and suggestions are made about some of the molecular evente believed to be responsible for the appearance of the five rate processes. For example, for DNA, poly[d(DAP-A-T)], and poly[d(DAP-T)], the observed order of apparent second-order rate constants, normalized to the concentration of actinomycin binding sites, suggests that binding of the antibiotic occurs most rapidly at binding sites (G-C of d DAT-T) near d(A-T) base pairs, where weakening of the double-helical conformation requires the least energy. Results obtained from studies of actinomycin D binding to heat-denatured poly[d(DAP-A-T)] and of actinomine and actinomycin D lactam binding to DNA suggest that the slow rate processes are related to an actinomycyl-pentapeptide-induced unwinding of the sugar-phosphate backbone of DNA accompanying insertion of the cyclic peptides into DNA.     

Charge distribution in 7-methylguanine regarding cation-pi interaction with protein factor eIF4E

Electric charge distribution in mRNA 5' cap terminus has been exhaustively characterized in respect to the affinity for cap-binding proteins. Formation of the stacked configuration of positively charged 7-methylguanine in between two aromatic amino acid rings, known as sandwich cation-pi stacking, is thought to be prerequisite for the specific recognition of the cap by eukaryotic initiation factor eIF4E; i.e., discrimination between the cap and nucleotides without the methyl group at N(7). Nuclear magnetic resonance spectroscopy of (15)N/(13)C-double-labeled 7-methylguanosine 5'-triphosphate and 7-methylguanosine, as well as their unsubstituted counterparts, GTP and guanosine, yielded characteristic changes of the electron-mediated spin-spin couplings and chemical shifts due to the methylation at N(7). The experimentally measured changes of the nuclear magnetic resonance parameters have been analyzed in respect to the electric charge distribution calculated by means of quantum chemical methods, and interpreted in terms of new proposed positive charge localization in the 7-methylguanine five-member ring.     

Stopped-flow studies of 2'-deoxynucleotide binding to thymidylate synthase

The mechanism of 2'-deoxynucleotide binding to Lactobacillus casei thymidylate synthase was studied using stopped-flow kinetic techniques to monitor the decrease in intrinsic protein fluorescence upon complex formation. The data were consistent with a two-step mechanism involving a rapid preequilibrium step to form the enzyme-2'-deoxynucleotide complex followed by a slow isomerization step. Rate and equilibrium constants were determined for the three 2'-deoxynucleotides (2'-deoxyuridylate, 2'-deoxythymidylate, and 5-fluoro-2'-deoxyuridylate) as a function of temperature. Similar free energy changes were found for all 2'-deoxynucleotides; however, the enthalpy and entropy changes for each step of the reaction differed for each 2'-deoxynucleotide. The thermodynamic profiles indicated that the isomerization step stabilized the enzyme-2'-deoxynucleotide complex by an additional 1500 cal/mol.     

Phosphorylation dynamics of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) is discordant with its potential to interact with eukaryotic initiation factor 4E (eIF4E)

Mammalian target of rapamycin (mTOR) controls cellular growth and proliferation by virtue of its ability to regulate protein translation. Eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) — a key mTOR substrate, binds and sequesters eIF4E to impede translation initiation that is supposedly overcome upon 4E-BP1 phosphorylation by mTOR. Ambiguity surrounding the precise identity of mTOR regulated sites in 4E-BP1 and their invariable resistance to mTOR inactivation raises concerns about phospho-regulated model proposed for 4E:4E-BP1 interaction. Our attempt to mimic dephosphorylation associated with rapamycin response by introducing phospho deficient mutants for sites implicated in regulating 4E:4E-BP1 interaction individually or globally highlighted no obvious difference in the quantum of their association with CAP bound 4E when compared with their phosphomimicked counterparts or the wild type 4E-BP1. TOS or RAIP motif deletion variants compromised for raptor binding and resultant phosphodeficiency did little to influence their association with CAP bound 4E. Interestingly ectopic expression of ribosomal protein S6 kinase 1 (S6K1) that restored 4E-BP1 sensitivity to rapamycin/Torin reflected by instant loss of 4E-BP1 phosphorylation, failed to bring about any obvious change in 4E:4E-BP1 stoichiometry. Our data clearly demonstrate a potential disconnect between rapamycin response of 4E-BP1 and its association with CAP bound 4E.     

The effects of E7 and E11 mutations on the kinetics of ligand binding to R state human hemoglobin

Association and dissociation rate constants for O2, CO, and methyl isocyanide binding to native and distal pocket mutants of R state human hemoglobin were measured using ligand displacement and partial photolysis techniques. Individual rate constants for the alpha and beta subunits were resolved by comparisons between the kinetic behavior of the native and mutant proteins. His-E7 was replaced with Gly and Gln in both alpha and beta subunits and with Phe in beta subunits alone. In separate experiments Val-E11 was replaced with Ala, Leu, and Ile in each globin chain. The parameters describing ligand binding to R state alpha subunits are sensitive to the size and polarity of the amino acids at positions E7 and E11. The distal histidine in this subunit inhibits the bimolecular rate of binding of both O2 and CO, sterically hinders bound CO and methyl isocyanide, and stabilizes bound O2 by hydrogen bonding. The Val-E11 side chain in alpha chains also appears to be part of the kinetic barrier to O2 and CO binding since substitution with Ala causes approximately 10-fold increases in the association rate constants for the binding of these diatomic ligands. However, substitution of Val-E11 by Ile produces only small decreases in the rates of ligand binding to alpha subunits. For R state beta subunits, the bimolecular rates of O2 and CO binding are intrinsically large, approximately 2-5-fold greater than those for alpha subunits, and with the exception of Val-E11----Ile mutation, little affected by substitutions at either the E7 or E11 positions. For the beta Val-E11----Ile mutant the association rate and equilibrium constants for all three ligands decreased 10-50-fold. All of these results agree with Shaanan's conclusions that the distal pocket in liganded beta subunits is more open whereas in alpha subunits bound ligands are more sterically hindered by adjacent distal residues (Shaanan, B. (1983) J. Mol. Biol. 171, 31-59). In the case of O2 binding to alpha subunits, the unfavorable steric effects are compensated by the formation of a hydrogen bond between the nitrogen atom of His-E7 and bound dioxygen.     

Effects of pH on kinetics of binding of mRNA-cap analogs by translation initiation factor eIF4E

Stopped-flow spectrofluorimetry and a theoretical method for predicting protonation equilibria in polyelectrolytes were combined in an analysis of the pH dependence of the kinetics of binding of analogues of the 5'-mRNA cap to the cap binding protein eIF4E. The computer simulations and available experimental data indicate that there are two titratable groups in the binding site of the protein and two titratable groups on the ligands directly involved in the binding, in addition to stacking interactions described by other groups. The observed pH dependencies of the rate constants obtained from the stopped-flow experiments are consistent with this finding. In particular, it is concluded that binding of both forms of the cap analogs regarding protonation at the N1 position of the guanine ring is efficient, and the shift to a predominantly protonated form of the ring takes place after formation of the complex.     

Stopped-flow studies of the kinetics of single-stranded DNA binding and wrapping around the Escherichia coli SSB tetramer

We have examined the kinetic mechanism for binding of the homotetrameric Escherichia coliSSB protein to single-stranded oligodeoxynucleotides [(dT)(70) and (dT)(35)] under conditions that favor the formation of a fully wrapped ssDNA complex in which all four subunits interact with DNA. Under these conditions, a so-called (SSB)(65) complex is formed in which either one molecule of (dT)(70) or two molecules of (dT)(35) bind per tetramer. Stopped-flow studies monitoring quenching of the intrinsic SSB Trp fluorescence were used to examine the initial binding step. To examine the kinetics of ssDNA wrapping, we used a single-stranded oligodeoxythymidylate, (dT)(66), that was labeled on its 3'-end with a fluorescent donor (Cy3) and on its 5'-end with a fluorescent acceptor (Cy5). Formation of the fully wrapped structure was accompanied by extensive fluorescence resonance energy transfer (FRET) from Cy3 to Cy5 since the two ends of (dT)(66) are in close proximity in the fully wrapped complex. Our results indicate that initial ssDNA binding to the tetramer is very rapid, with a bimolecular rate constant, k(1,app), of nearly 10(9) M(-1) s(-1) in the limit of low salt concentration (<0.2 M NaCl, pH 8.1, 25.0 degrees C), whereas the rate of dissociation is very low at all salt concentrations that were examined (20 mM to 2 M NaCl or NaBr). However, the rate of initial binding and the rate of formation of the fully wrapped complex are identical, indicating that the rate of wrapping of the ssDNA around the SSB tetramer is very rapid, with a lower limit rate of 700 s(-1). The implications of this rapid binding and wrapping reaction are discussed.     

Phosphorylation of the mRNA cap-binding protein eIF4E and cancer

Dysregulated protein synthesis is frequently involved in oncogenesis and cancer progression. Translation initiation is thought to be the rate-limiting step in protein synthesis, and the mRNA 5′ cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) is a pivotal factor that initiates translation. The activities of eIF4E are regulated at multiple levels, one of which is through its phosphorylation at Serine 209 by the mitogen-activated protein kinase-interacting kinases (MNKs, including MNK1 and MNK2). Benefiting from novel mouse genetic tools and pharmacological MNK inhibitors, our understanding of a role for eIF4E phosphorylation in tumor biology and cancer therapy has greatly evolved in recent years. Importantly, recent studies have found that the level of eIF4E phosphorylation is frequently upregulated in a wide variety of human cancer types, and phosphorylation of eIF4E drives a number of important processes in cancer biology, including cell transformation, proliferation, apoptosis, metastasis and angiogenesis. The MNK-eIF4E axis is being assessed as a therapeutic target either alone or in combination with other therapies in different cancer models. As novel MNK inhibitors are being developed, experimental studies bring new hope to cure human cancers that are not responsive to traditional therapies. Herein we review recent progress on our understanding of a mechanistic role for phosphorylation of eIF4E in cancer biology and therapy.     

A second eIF4E protein in Schizosaccharomyces pombe has distinct eIF4G-binding properties

The eukaryotic cap-binding proteins belonging to the eIF4E family are generally involved in mediating the recruitment of ribosomes to capped mRNA. We described previously a cap-binding protein (now called eIF4E1) in Schizosaccharomyces pombe that appears to have all of the usual structural and functional attributes of an eIF4E. We have now characterised a new type of cap-binding protein (eIF4E2) from this organism, which at the amino acid sequence level, is 52% identical and 59% similar to eIF4E1. eIF4E2 is not essential in S.pombe but has some novel properties that may be related to a special function in the cell. The ratio of eIF4E2:eIF4E1 in the cell shifts in favour of eIF4E2 at higher temperatures. Despite having all of the dorsal face amino acids that have so far been associated with eIF4G binding to eIF4E1, eIF4E2 binds the eIF4E-binding domain of S.pombe eIF4G >10²-times weaker than eIF4E1 in vitro. The eIF4E2 cap-binding affinity is in the typical micromolar range. The results suggest that eIF4E2 is not active on the main pathway of translation initiation in fission yeast but might play a role in the adaptation strategy of this organism under specific growth conditions. Moreover, they provide insight into the molecular characteristics required for tight binding to eIF4G.