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Discrimination between mono- and trimethylated cap structures by two isoforms of Caenorhabditis elegans eIF4E

2002; Springer Nature; Volume: 21; Issue: 17 Linguagem: Inglês

10.1093/emboj/cdf473

1460-2075

Hiroshi Miyoshi,

Enzyme Catalysis and Immobilization

Article2 September 2002free access Discrimination between mono–and trimethylated cap structures by two isoforms of Caenorhabditis elegans eIF4E Hiroshi Miyoshi Hiroshi Miyoshi Present address: Genomics Research Institute, Utsunomiya University, Utsunomiya-Shi, 321-8505 Japan Search for more papers by this author Donard S. Dwyer Donard S. Dwyer Department of Psychiatry, Louisiana State University Health Sciences Center, Shreveport, LA, 71130-3932 USA Search for more papers by this author Brett D. Keiper Brett D. Keiper Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA, 71130-3932 USA Search for more papers by this author Marzena Jankowska-Anyszka Marzena Jankowska-Anyszka Department of Chemistry, University of Warsaw, 02-093 Warsaw, Poland Search for more papers by this author Edward Darzynkiewicz Edward Darzynkiewicz Department of Biophysics, University of Warsaw, 02-093 Warsaw, Poland Search for more papers by this author Robert E. Rhoads Corresponding Author Robert E. Rhoads Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA, 71130-3932 USA Search for more papers by this author Hiroshi Miyoshi Hiroshi Miyoshi Present address: Genomics Research Institute, Utsunomiya University, Utsunomiya-Shi, 321-8505 Japan Search for more papers by this author Donard S. Dwyer Donard S. Dwyer Department of Psychiatry, Louisiana State University Health Sciences Center, Shreveport, LA, 71130-3932 USA Search for more papers by this author Brett D. Keiper Brett D. Keiper Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA, 71130-3932 USA Search for more papers by this author Marzena Jankowska-Anyszka Marzena Jankowska-Anyszka Department of Chemistry, University of Warsaw, 02-093 Warsaw, Poland Search for more papers by this author Edward Darzynkiewicz Edward Darzynkiewicz Department of Biophysics, University of Warsaw, 02-093 Warsaw, Poland Search for more papers by this author Robert E. Rhoads Corresponding Author Robert E. Rhoads Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA, 71130-3932 USA Search for more papers by this author Author Information Hiroshi Miyoshi2, Donard S. Dwyer3, Brett D. Keiper1, Marzena Jankowska-Anyszka4, Edward Darzynkiewicz5 and Robert E. Rhoads 1 1Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA, 71130-3932 USA 2Present address: Genomics Research Institute, Utsunomiya University, Utsunomiya-Shi, 321-8505 Japan 3Department of Psychiatry, Louisiana State University Health Sciences Center, Shreveport, LA, 71130-3932 USA 4Department of Chemistry, University of Warsaw, 02-093 Warsaw, Poland 5Department of Biophysics, University of Warsaw, 02-093 Warsaw, Poland *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:4680-4690https://doi.org/10.1093/emboj/cdf473 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Primitive eukaryotes like Caenorhabditis elegans produce mRNAs capped with either m7GTP or m32,2,7GTP. Caenorhabditis elegans also expresses five isoforms of the cap-binding protein eIF4E. Some isoforms (e.g. IFE-3) bind to m7GTP–Sepharose exclusively, whereas others (e.g. IFE-5) bind to both m7GTP− and m32,2,7GTP–Sepharose. To examine specificity differences, we devised molecular models of the tertiary structures of IFE-3 and IFE-5, based on the known structure of mouse eIF4E-1. We then substituted amino acid sequences of IFE-5 with homologous sequences from IFE-3. As few as two changes (N64Y/V65L) converted the cap specificity of IFE-5 to essentially that of IFE-3. Molecular dynamics simulations suggested that the width and depth of the cap-binding cavity were larger in IFE-5 than in IFE-3 or the N64Y/V65L variant, supporting a model in which IFE-3 discriminates against m32,2,7GTP by steric hindrance. Furthermore, the affinity of IFE-5 (but not IFE-3) for m32,2,7GTP was reversibly increased when thiol reagents were removed. This was correlated with the formation of a disulfide bond between Cys-122 and Cys-126. Thus, translation of m32,2,7GTP-capped mRNAs may be regulated by intracellular redox state. Introduction Initiation of protein synthesis proceeds by progressive assembly of initiation complexes, each stage catalyzed by a different set of initiation factors (Hershey and Merrick, 2000). Recruitment of mRNA to the 43S initiation complex in eukaryotes requires the eIF4 factors, which include the 25 kDa cap-binding protein eIF4E. Alterations in eIF4E levels and activity have a profound effect on cell growth and phenotype, presumably due to the differential recruitment of mRNAs specifically required for cell growth and cell cycle progression (De Benedetti and Harris, 1999). The tertiary structure of mouse eIF4E-1 was solved by X-ray crystallography (Marcotrigiano et al., 1997) and that of yeast eIF4E by NMR spectroscopy (Matsuo et al., 1997). The binding of m7GDP results from the stacking of the purine ring between Trp-56 and Trp-102 (using the mouse numbering), formation of H-bonds between N1 and N2 of m7G and Glu-103, van der Waals interactions between the ribose moiety and Trp-56, and ionic interactions of the α- and β-phosphate oxygen atoms with Arg-157 and Lys-162. Addition of one methyl group at the N2 position of m7G has little effect on cap binding to mammalian eIF4E, but addition of a second methyl group, to form 2,2,7-trimethylguanine (m32,2,7G), markedly decreases binding (Darzynkiewicz et al., 1988; Carberry et al., 1990; Cai et al., 1999). Two types of capped RNAs exist in eukaryotic cells. The primary transcripts for both mRNAs and small nuclear RNAs (snRNAs) are modified to form a 5′–5′ GpppN linkage, which is subsequently methylated in the nucleus to yield m7GpppN (Varani, 1997). The cap of snRNAs is further methylated in the cytosol at N2 of m7G, forming m32,2,7GpppN (Mattaj, 1986). These methylations are dependent upon the binding of Sm proteins to form U-type small nuclear ribonuclear protein complexes (snRNPs) and are the signal for import of snRNPs into the nucleus (Hamm et al., 1990; Gorlich and Mattaj, 1996). The trimethylated cap structure is recognized by Snurportin1, a receptor for spliceosomal mRNPs that utilizes the importin β pathway for nuclear import (Huber et al., 1998). The strong preference of most eIF4Es for m7G- versus m32,2,7G-containing caps ensures that mRNAs rather than snRNAs are recruited to the translational machinery. An exception to this paradigm is the acquisition of m32,2,7G-containing caps by mRNAs through the process of trans-splicing, in which a 22 nucleotide spliced leader is transferred from the 5′-end of an snRNA to an acceptor site in the 5′ end of the pre-mRNA (Blumenthal, 1998). As a result, the original m7G-containing cap is replaced by the m32,2,7G-containing cap (van Doren and Hirsh, 1990). Trans-splicing of mRNA has been most studied in primitive eukaryotes like the nematode Caenorhabditis elegans (Zorio et al., 1994), but it also occurs in more complex chordate species (Vandenberghe et al., 2001). In C.elegans, the majority of mRNAs are trans-spliced. Both m7G- and m32,2,7G-capped mRNAs are found in the cytosol of C.elegans and are translated on polyribosomes (Liou and Blumenthal, 1990). The fact that the well-studied eIF4Es of higher eukaryotes strongly prefer m7G- to m32,2,7G-capped mRNAs, yet C.elegans translates both types of mRNAs, led us to examine the eIF4E of C.elegans (Jankowska-Anyszka et al., 1998; Keiper et al., 2000). Surprisingly, five eIF4E isoforms are expressed in C.elegans, named IFE-1 to IFE-5. IFE-3 is the most homologous to mammalian eIF4E-1, is retained on m7GTP–Sepharose but not m32,2,7GTP–Sepharose, and is the only IFE that is essential for viability. IFE-4 has the same cap specificity, but is not essential. By contrast, two isoforms with closely related amino acid sequences, IFE-1 and IFE-5, are retained on both m7GTP–Sepharose and m32,2,7GTP–Sepharose. IFE-2, while not retained on m32,2,7GTP–Sepharose, is nonetheless prevented from binding to m7GTP–Sepharose by m32,2,7GTP. IFE-1, -2 and -5 are partially redundant for viability, but at least one isoform is required. IFE-1 is expressed only in the gonad of maturing worms and plays an essential role for spermatogenesis in both males and hermaphrodites (Amiri et al., 2001). In the present study, we have examined the structural basis for differences in cap discrimination among the IFE proteins. Such knowledge may give insight into the biological roles of the five IFE proteins of C.elegans, as well as the multiple eIF4E isoforms found in other organisms (Browning et al., 1987; Rychlik et al., 1987; Wakiyama et al., 1995; Gao et al., 1998; Myers et al., 2000). We find that changing just two amino acid residues can alter the cap discrimination of IFE-5 to resemble that of IFE-3. Based on dynamic tertiary structure models, we propose that this change results from a reduction in the average size of the cap-binding cavity. Un expectedly, the cap discrimination of IFE-5 is reversibly changed by the formation of a disulfide bond between two Cys residues. Results Rationale and approach for determining the basis for cap discrimination It is possible that a small subset of amino acid residues is responsible for the difference in cap specificity of the two classes of C.elegans eIF4E isoforms. Our broad strategy was to choose a representative of each eIF4E class, IFE-3 and IFE-5, interchange amino acid residues, and examine the specificity of cap recognition. One way to choose candidates for discriminatory amino acid residues is to examine regions that are likely to be in close proximity to the bound cap structure. Unfortunately, the tertiary structure has not been solved for any of the IFE proteins. However, the co-crystal structure of mouse eIF4E with m7GDP has been solved and can serve as a template for model building of the IFE proteins. Amino acid sequence alignment shows that mouse eIF4E is more similar to IFE-3 than to IFE-5 (Figure 1), in agreement with the calculated sequence identities (47% versus 41%) and the strong preference of both mammalian eIF4E and IFE-3 for m7G-containing caps (Jankowska-Anyszka et al., 1998; Cai et al., 1999). Importantly, there is a high degree of sequence identity between mouse eIF4E and both IFE-3 and IFE-5 in the β-sheets identified by X-ray crystallography (s1, s3, etc.). We approximated the tertiary structures for IFE-3 and IFE-5 by homology modeling to the mouse eIF4E structure (Dwyer, 1996, 2001; Figure 2). After energy minimization, the root-mean-square deviations are 2.2 and 1.1 Å, respectively. The residues homologous to Trp-56 and Trp-102 of mouse eIF4E, which 'sandwich' the guanine ring, are Trp-51 and Trp-97 in IFE-3, and Trp-28 and Trp-74 in IFE-5. Figure 1.Sequence comparisons between C.elegans IFE-3, mouse eIF4E and C.elegans IFE-5. Amino acid sequences were deduced from the cDNA sequences of IFE-3 (Jankowska-Anyszka et al., 1998), mouse eIF4E (Altmann et al., 1989) and IFE-5 (Keiper et al., 2000). Alignment was performed using the CLUSTAL W algorithm (version 1.8; http://www.ddbj.nig.ac.jp). Shading indicates identical residues. Secondary structure elements are designated as follows: S1, β-sheet 1; S2, β-sheet 2; H1, α-helix 1; H2, α-helix 2; etc. Amino acid residues that contact the cap in mouse eIF4E are shown in bold. Download figure Download PowerPoint Figure 2.Molecular models of IFE-3, IFE-5 and the NV-YL variant of IFE-5 in comparison with the crystal structure of mouse eIF4E. The figure was produced with the graphics interface of the Insight II software. N-terminal amino acid residues homologous to those truncated from mouse eIF4E prior to X-ray crystallography are not shown. The position of m7GDP is shown in the mouse eIF4E structure. The structures of IFE-3, IFE-5 and IFE-5(NV-YL) were obtained from equivalent time points in MD simulations and are intended to show dynamic aspects of the binding site rather than the minimized structures. Selected amino acid side chains are shown in different colors and labeled in upper case letters using the one-letter code. Secondary structure elements (β-sheets in yellow, loops in dark blue, α-helices in light blue) are labeled in lower case letters, e.g. β-sheet 3 is 's3', etc. Loops connecting β-sheets are labeled 1–2 loop, etc. IFE structures are oriented the same as mouse eIF4E. Download figure Download PowerPoint The similarity of the IFE-3 and IFE-5 models (Figure 2), coupled with a knowledge of where the amino acid sequences deviate (Figure 1), allowed us to choose amino acid sequences that were likely to be involved in cap discrimination. The loop connecting β-sheets 1 and 2, referred to as the 1–2 loop, forms one side of the cap-binding pocket and could contribute side chains that determine specificity. The 3–4 loop, connecting β-sheets 3 and 4, forms the other side of the cap-binding pocket. It also contains a Glu residue homologous to Glu-103 in mouse eIF4E, which forms H-bonds with the N1 and N2 protons of m7G; these protons are absent in m32,2,7G. The loops are, therefore, attractive sites for modification. Replacement of amino acid sequences in IFE-5 with homologous sequences from IFE-3 In variant 1–2 loop, the 1–2 loop in IFE-5 is replaced by the equivalent sequence from IFE-3 (Figure 3). This loop contains one of the two Trp residues sandwiching the cap guanine. In variant 3–4 up, the upstream (N-terminal) half of the 3–4 loop is replaced together with part of β-sheet 3, whereas in variant 3–4 down, the downstream (C-terminal) half of this same loop is replaced. This stretch of amino acids includes the other Trp residue sandwiching the m7G. In other variants, the entire 3–4 loop is replaced (3–4 loop), or various types of 1–2 and 3–4 loop substitutions are combined (1–2 & 3–4 up, 1–2 & 3–4 down, and 1–2 & 3–4 loop). Finally, the upstream portion of the 3–4 loop is dissected with more selective substitutions (variants N-Y, V-L, NV-YL, DDIQPK-EGIKPM and QPK-KPM). Figure 3.Amino acid substitutions in IFE-5. Variants, named on the left, were constructed by substituting the indicated sequences of IFE-3 into the homologous positions of IFE-5. Letters in bold indicate amino acid residues that are conserved between IFE-3 and IFE-5. Download figure Download PowerPoint Qualitative testing of variants using affinity chromatography IFE-5 variants were initially screened qualitatively for cap specificity by measuring their capture from Escherichia coli lysates on columns of either m7GTP− or m32,2,7GTP–Sepharose, followed by elution with m7GTP or m32,2,7GTP, respectively (Figure 4). In this qualitative assay, it is not possible to distinguish between proteins that are more highly expressed in soluble form and those with enhanced cap-binding activity; this distinction is made by measuring cap affinities of purified IFEs using the fluorescence-quenching assay (see below). Rather, the qualitative assays served to guide the construction of IFE-5 variants that showed a change in the relative binding to m7GTP–Sepharose versus m32,2,7GTP–Sepharose compared with wild-type (wt) IFE-5. Figure 4.Qualitative testing of IFE-5 sequence variants using affinity chromatography. Escherichia coli was transfected with the indicated expression vectors, and extracts were applied to columns of m7GTP–Sepharose (M) or m32,2,7GTP–Sepharose (T). Proteins were eluted with m7GTP or m32,2,7GTP, respectively, and analyzed by SDS–PAGE, with Coomassie Blue staining. Download figure Download PowerPoint As reported previously (Jankowska-Anyszka et al., 1998), IFE-3 is retained on m7GTP–Sepharose (Figure 4A, lane M under IFE-3) but not m32,2,7GTP–Sepharose (lane T). This indicates high selectivity for m7G-containing caps, i.e. a 'mono-specific' IFE. By contrast, IFE-5 is retained both by m7GTP–Sepharose (Figure 4A, lane M under IFE-5) and, to a lesser extent, by m32,2,7GTP–Sepharose (lane T). This is the behavior of a 'dual-specific' IFE. Comparing wt IFE-5 to several variants for relative binding to m7GTP–Sepharose versus m32,2,7GTP–Sepharose, variant 3–4 up appears to show the highest selectivity for m7GTP (Figure 4A). The variant 3–4 down, on the other hand, binds m32,2,7GTP–Sepharose more efficiently than wt IFE-5, despite similar binding to m7GTP–Sepharose. Thus, it displays lower selectivity for m7GTP than IFE-5. These results suggest that a discriminatory element (favoring m7GTP over m32,2,7GTP) exists in the upstream but not the downstream portion of the 3–4 loop. We next explored combinations of 1–2 loop sequences with 3–4 loop sequences. Variant 1–2 & 3–4 down is more selective for m7GTP than the corresponding variant 3–4 down (Figure 4A). Variant 1–2 & 3–4 up (Figure 4B), on the other hand, is less selective than the corresponding variant 3–4 up. Similarly, variant 1–2 & 3–4 loop is less selective than variant 3–4 loop. In all three cases, the changes in cap selectivity caused by substitutions in the 3–4 loop are attenuated by substitutions in the 1–2 loop, causing the proteins to behave more like wt IFE-5. Based on these findings, we sought the specific amino acid residues in the upper 3–4 loop sequence responsible for increased m7GTP selectivity. Of several single and double amino acid substitutions, the variant NV-YL produces the highest selectivity: binding to m32,2,7GTP–Sepharose is nearly undetectable despite robust binding to m7GTP–Sepharose (Figure 4C and D). The combination of changing both Asn-64 and Val-65, rather than either alone, confers the maximum change in m7GTP selectivity. We explored the remaining portion of the 3–4 up region with the variant DDIQPK-EGIKPM (Figure 4E). Despite the fact that binding to both affinity resins is lower than for wt IFE-5, the ratio of binding appears similar to that of wt IFE-5, suggesting no change in selectivity. By contrast, the variant QPK-KPM displays increased binding to m32,2,7GTP–Sepharose, but the same or even less binding to m7GTP–Sepharose than IFE-5 (Figure 4F). This indicates that QPK-KPM is even more permissive than IFE-5 for m32,2,7GTP binding. A consistent picture emerges from these observations. The 1–2 loop region alone does not affect m7GTP selectivity. By contrast, the 3–4 loop region is responsible for m7GTP selectivity, but changes in selectivity are 'damped' in variants combining 1–2 loop and 3–4 loop substitutions. Within the 3–4 loop region, the N-terminal portion (3–4 up) increases selectivity, while the C-terminal portion decreases selectivity. Within the 3–4 up region, simultaneously replacing Asn-64 with Tyr and Val-65 with Leu confers the greatest selectivity, indistinguishable from IFE-3 by this assay. The individual substitutions at positions 64 or 65 do not confer this degree of enhanced selectivity, nor do substitutions in the C-terminal portion of the 3–4 up region. In fact, replacement of the C-terminal one-third of the 3–4 up region has the opposite effect, lowering selectivity. This may account for the observation that exchanging 64NV65 (variant NV-YL) increases selectivity more than exchanging 64NVFRDDIQPK73 (variant 3–4 up). Quantitative testing of 1–2 and 3–4 loop IFE-5 variants by fluorescence quenching To obtain quantitative binding data, we employed the fluorescence-quenching assay with homogeneous IFE proteins. The dissociation constants of the cap analog· protein complexes, KD, were calculated for both m7GTP and m32,2,7GTP titrations (Table I, Experiment 1). The binding of m7GTP to IFE-3 is 1.3-fold stronger than to IFE-5 (KD = 0.38 versus 0.50 μM). By contrast, the binding of m32,2,7GTP is 2.3-fold stronger to IFE-5 than to IFE-3 (KD = 1.6 versus 3.5 μM). The combined effects of these nucleotide-binding differences is that IFE-3 discriminates in favor of m7GTP versus m32,2,7GTP caps by a factor of 9.2, whereas IFE-5 discriminates by a factor of only 3.2 (Table I, KD ratio). Table 1. Dissociation constants (KD) for m7GTP and m32,2,7GTP from various IFE proteins Proteina Titration bufferb KD (μM)c KD ratiod m7GTP m32,2,7GTP Experiment 1 IFE-3 A1 0.38 ± 0.03 3.5 ± 0.1 9.2 ± 0.8 IFE-5 A1 0.50 ± 0.06 1.6 ± 0.1 3.2 ± 0.6 1–2 loop A1 0.35 ± 0.02 1.5 ± 0.1 4.3 ± 0.5 3–4 up A1 0.43 ± 0.03 1.9 ± 0.1 4.4 ± 0.5 3–4 down A1 0.41 ± 0.03 0.81 ± 0.02 2.0 ± 0.2 3–4 loop A1 0.46 ± 0.03 1.9 ± 0.1 4.1 ± 0.5 1–2 & 3–4 up A1 0.55 ± 0.05 1.5 ± 0.1 2.7 ± 0.4 1–2 & 3–4 down A1 0.46 ± 0.02 1.4 ± 0.1 3.0 ± 0.3 1–2 & 3–4 loop A1 0.42 ± 0.03 1.1 ± 0.1 2.6 ± 0.4 N-Y A1 0.41 ± 0.02 1.4 ± 0.1 3.4 ± 0.4 V-L A1 0.43 ± 0.04 2.3 ± 0.1 5.3 ± 0.7 NV-YL A1 0.38 ± 0.03 2.6 ± 0.1 6.8 ± 0.8 DDIQPK-EGIKPM A1 0.51 ± 0.04 1.6 ± 0.1 3.1 ± 0.4 QPK-KPM A1 0.45 ± 0.03 0.85 ± 0.04 1.9 ± 0.2 Experiment 2 IFE-3 A0 0.36 ± 0.03 3.5 ± 0.1 9.7 ± 1.1 IFE-5 A0 0.54 ± 0.07 0.92 ± 0.08 1.7 ± 0.4 3–4 up A0 0.47 ± 0.03 0.94 ± 0.03 2.0 ± 0.2 3–4 loop A0 0.45 ± 0.04 0.95 ± 0.04 2.1 ± 0.3 NV-YL A0 0.68 ± 0.03 1.2 ± 0.1 1.8 ± 0.2 QPK-KPM A0 0.57 ± 0.03 1.0 ± 0.1 1.8 ± 0.3 IFE-3 A10 0.39 ± 0.03 3.5 ± 0.1 9.0 ± 0.9 IFE-5 A10 0.48 ± 0.05 1.9 ± 0.1 4.0 ± 0.6 NV-YL A10 0.32 ± 0.04 2.7 ± 0.1 8.4 ± 1.6 a Proteins were dialyzed against buffer A1 in Experiment 1 and against buffer A0 in Experiment 2 prior to titration. Buffer A0 is 50 mM HEPES pH 7.6, 1 mM EDTA, 50 mM KCl, 5% glycerol. Buffer A1 is buffer A0 containing 1 mM DTT. b Titrations were carried out in the indicated buffers. Buffer A10 is buffer A0 containing 10 mM DTT. c KD values were determined by measuring the quenching of intrinsic Trp fluorescence during titration with the indicated cap analogs. d The KD obtained with m3GTP was divided by the KD obtained with mGTP. The quantitative assay confirmed that substitutions of IFE-3 sequences into IFE-5 change m7GTP selectivity. Variants 1–2 loop, 3–4 loop and 3–4 up are slightly more selective than wt IFE-5 (increase in KD ratio), whereas 3–4 down is less selective. However, the largest increase is observed with variant NV-YL (KD ratio = 6.8), which discriminates nearly as well as IFE-3 (KD ratio = 9.2). The change in selectivity for variant NV-YL occurs because of both an increase in affinity for m7GTP (1.3-fold) and a decrease in affinity for m32,2,7GTP (1.7-fold). Interestingly, neither the N-Y nor V-L variants alone show as much increase in KD ratio as the combination. Some variants are less selective for m7GTP than wt IFE-5, as reflected in a lower KD ratio. The largest effect is for variant QPK-KPM, which has a KD ratio of 1.9 compared with 3.2 for wt IFE-5. This change results from an increase in m32,2,7GTP affinity rather than a decrease in m7GTP affinity. Change in selectivity as a function of redox state A preliminary tertiary structure for IFE-5 revealed a close proximity of Cys-122 and Cys-142, suggesting the possibility of a disulfide bond between them. Also, the two proteins retained on m32,2,7GTP–Sepharose (IFE-1 and IFE-5) contain Cys residues at four homologous positions, Cys-61, Cys-126, Cys-142 and Cys-185 (using the IFE-5 numbering), that are not present in IFE-3. Conceivably, the tertiary structure of IFE-5 is governed, in part, by disulfide bonds. We therefore repeated the quantitative determination of KD values in the absence of reducing agent. Titration of IFE-5 with m7GTP in buffer A0, which does not contain dithiothreitol (DTT), reveals a KD for m7GTP of 0.54 μM (Table I, Experiment 2). This is statistically the same as the value obtained in buffer A1, 0.50 μM (Experiment 1). By contrast, titration with m32,2,7GTP in buffer A0 yields a KD of 0.92 μM, compared with 1.6 μM in buffer A1. Thus, the affinity of IFE-5 for m32,2,7GTP increases in the absence of DTT. The overall effect is a 1.9-fold loss of m7GTP selectivity under more oxidizing conditions (KD ratio decreases from 3.2 in buffer A1 to 1.7 in buffer A0). For variants 3–4 up and 3–4 loop, the affinity for m7GTP is statistically the same in the presence or absence of DTT, but the affinity for m32,2,7GTP increases in buffer A0. This leads to an overall decrease in m7GTP selectivity of 2.0- to 2.2-fold for these two variants. The results are even more dramatic for variant NV-YL; the KD values obtained in buffer A0 indicate a loss of affinity for m7GTP and a gain of affinity for m32,2,7GTP compared with buffer A1, for a loss of m7GTP selectivity of 3.8-fold. Overall, the KD ratios of IFE-5 and all variants tested decreased to approximately the same value in buffer A0 (average of 2.2 ± 0.6), whereas they ranged as high as 6.8 ± 0.8 in buffer A1. We tested the specificity of this effect with several controls. First, the selectivity of IFE-3 is statistically unchanged in buffer A0 (KD ratio = 9.7 ± 0.8; Experiment 2) versus buffer A1 (KD ratio = 9.2 ± 1.1; Experiment 1). Second, the proteins that had been dialyzed against buffer A0 were titrated with cap analogs in buffer A10 (Experiment 2). The KD values for IFE-3 are all statistically the same in buffer A0, A1 or A10. However, the KD values and ratios for IFE-5 and variant NV-YL revert to their pre-dialysis values. The fact that these proteins recover their high selectivity for m7GTP in the presence of DTT shows that subjecting them to air oxidation (dialysis against buffer A0) does not result in protein denaturation. As a third control, we tested whether redox state influences overall protein stability. IFE-3 and IFE-5, previously dialyzed against buffer A0, were subjected to progressively higher concentrations of guanidine isothiocyanate (0–2.67 M) in the presence or absence of 10 mM DTT. The proportion of denatured protein was estimated from the ratio of fluorescence emission at 335 nm to that at 355 nm (Hammarström et al., 2001). The denaturation curves for both proteins were the same with or without DTT (data not shown), indicating that air oxidation does not affect gross IFE structure. Since DTT (Cleland, 1964) is a much stronger reducing agent than glutathione (GSH), the predominant reducing agent in the cell, we considered the possibility that the change in cap specificity induced by 1 mM DTT occurs outside the range of normal intracellular reduction potentials. We therefore repeated the titration of IFE-5 with m7GTP and m32,2,7GTP at five physiological combinations (Shan et al., 1990) of reduced and oxidized (GSSG) glutathione. The KD ratio was then determined for each redox condition (Figure 5). The results indicated that the affinities of IFE-5 for m7GTP and m32,2,7GTP are nearly equal (KD ratio = 1.3 ± 0.3) in 0 mM GSH and 2.5 mM GSSG. Selectivity increases as the GSH/GSSG ratio increases, reaching a maximum KD ratio of 2.6 ± 0.4. Figure 5.Change in cap specificity of IFE-5 as a function of reduction potential. The KD values for dissociation of m7GTP and m32,2,7GTP from IFE-5 were determined by fluorescence quenching in buffer A0 containing the indicated concentrations of reduced (GSH) and oxidized (GSSG) glutathione. The KD ratios were computed as described in Table I. Download figure Download PowerPoint Identification of a disulfide bond between Cys-122 and Cys-126 We sought direct evidence that IFE-5 undergoes reversible formation of disulfide bonds. Since the five Cys residues of IFE-5 occur in four different tryptic peptides, we adopted the following strategy. IFE-5 in either the oxidized or reduced states (i.e. dialyzed against either buffer A0 or A1) was alkylated with acrylamide, digested with trypsin and the peptides subjected to matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Peptides containing Cys residues in the sulfhydryl state will have masses that are 71.0 Da greater for every acrylamide moiety incorporated. Peptides containing Cys residues in the disulfide state will not react with acrylamide, and their masses will correspond to the peptide containing a sulfhydryl, an intrastrand disulfide, a mixed disulfide (with another peptide), or some combination of these. By comparing IFE-5 subjected to the same reducing or oxidizing conditions that produced the change in cap specificity, we tested whether the oxidation state of Cys residues could be correlated with the functional change. Figure 6 shows selected regions of the mass spectra for tryptic peptides from IFE-5 that had been subjected to acrylamidation in either the oxidized (Figure 6A and B) or reduced (Figure 6C and D) states. The peak in Figure 6C at 1580.4 Da is within 0.3 Da of the predicted monoisotopic mass for the doubly acrylamidated peptide 117DMESIC GLVCNVR129, which contains Cys-122 and Cys 126. (The peaks at 1581.4, 1582.4 and 1583.4 are isomers of the same peptide containing one, two or three 13C atoms, respectively.) Additional evidence that the cluster of peaks at m/z = 1580.4–1583.4 represents peptide 117DMESIC GLVCNVR129 comes from a second cluster of peaks at 1596.4–1599.4, representing the same peptides containing Met sulfoxide. The m/z = 1580.4–1583.4 peaks are present in much lower amounts for IFE-5 first dialyzed against buffer A0 (Figure 6A), indicating that Cys-122 and Cys-126 are predominantly in the disulfide form and therefore unable to react with acrylamide. The absence of a peak at m/z = 1509.4 in Figure 6A, which would have been produced by singly acrylamidated 117D