A conserved motif within the flexible C-terminus of the translational regulator 4E-BP is required for tight binding to the mRNA cap-binding protein eIF4E

Although the central α-helical Y(X)4LΦ motif (X, variable amino acid; Φ, hydrophobic amino acid) of the translational regulator 4E-BP [eIF (eukaryotic initiation factor) 4E-binding protein] is the core binding region for the mRNA cap-binding protein eIF4E, the functions of its N- and C-terminal flexible regions for interaction with eIF4E remain to be elucidated. To identify the role for the C-terminal region in such an interaction, the binding features of full-length and sequential C-terminal deletion mutants of 4E-BPn (n=1–3) subtypes were investigated by SPR (surface plasmon resonance) analysis and ITC (isothermal titration calorimetry). Consequently, the conserved PGVTS/T motif within the C-terminal region was shown to act as the second binding region and to play an important role in the tight binding to eIF4E. The 4E-BP subtypes increased the association constant with eIF4E by approximately 1000-fold in the presence of this conserved region compared with that in the absence of this region. The sequential deletion of this conserved region in 4E-BP1 showed that deletion of Val81 leads to a considerable decrease in the binding ability of 4E-BP. Molecular dynamics simulation suggested that the conserved PGVTS/T region functions as a kind of paste, adhering the root of both the eIF4E N-terminal and 4E-BP C-terminal flexible regions through a hydrophobic interaction, where valine is located at the crossing position of both flexible regions. It is concluded that the conserved PGVTS/T motif within the flexible C-terminus of 4E-BP plays an auxiliary, but indispensable, role in strengthening the binding of eIF4E to the core Y(X)4LΦ motif.

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Dissociation of the Tubulin-sequestering and Microtubule Catastrophe-promoting Activities of Oncoprotein 18/Stathmin

Molecular Biology of the Cell ◽

10.1091/mbc.10.1.105 ◽

1999 ◽

Vol 10 (1) ◽

pp. 105-118 ◽

Cited By ~ 131

Author(s):

Bonnie Howell ◽

Niklas Larsson ◽

Martin Gullberg ◽

Lynne Cassimeris

Keyword(s):

Tight Binding ◽

Terminal Region ◽

Microtubule Assembly ◽

Gtp Hydrolysis ◽

Truncated Protein ◽

C Terminus ◽

Dependent Change ◽

Tubulin Subunit ◽

Oncoprotein 18

Oncoprotein 18/stathmin (Op18) has been identified recently as a protein that destabilizes microtubules, but the mechanism of destabilization is currently controversial. Based on in vitro microtubule assembly assays, evidence has been presented supporting conflicting destabilization models of either tubulin sequestration or promotion of microtubule catastrophes. We found that Op18 can destabilize microtubules by both of these mechanisms and that these activities can be dissociated by changing pH. At pH 6.8, Op18 slowed microtubule elongation and increased catastrophes at both plus and minus ends, consistent with a tubulin-sequestering activity. In contrast, at pH 7.5, Op18 promoted microtubule catastrophes, particularly at plus ends, with little effect on elongation rates at either microtubule end. Dissociation of tubulin-sequestering and catastrophe-promoting activities of Op18 was further demonstrated by analysis of truncated Op18 derivatives. Lack of a C-terminal region of Op18 (aa 100–147) resulted in a truncated protein that lost sequestering activity at pH 6.8 but retained catastrophe-promoting activity. In contrast, lack of an N-terminal region of Op18 (aa 5–25) resulted in a truncated protein that still sequestered tubulin at pH 6.8 but was unable to promote catastrophes at pH 7.5. At pH 6.8, both the full length and the N-terminal–truncated Op18 bound tubulin, whereas truncation at the C-terminus resulted in a pronounced decrease in tubulin binding. Based on these results, and a previous study documenting a pH-dependent change in binding affinity between Op18 and tubulin, it is likely that tubulin sequestering observed at lower pH resulted from the relatively tight interaction between Op18 and tubulin and that this tight binding requires the C-terminus of Op18; however, under conditions in which Op18 binds weakly to tubulin (pH 7.5), Op18 stimulated catastrophes without altering tubulin subunit association or dissociation rates, and Op18 did not depolymerize microtubules capped with guanylyl (α, β)-methylene diphosphonate–tubulin subunits. We hypothesize that weak binding between Op18 and tubulin results in free Op18, which is available to interact with microtubule ends and thereby promote catastrophes by a mechanism that likely involves GTP hydrolysis.

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Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus

Molecular and Cellular Biology ◽

10.1128/mcb.9.1.83-91.1989 ◽

1989 ◽

Vol 9 (1) ◽

pp. 83-91

Author(s):

S Miyazawa ◽

T Osumi ◽

T Hashimoto ◽

K Ohno ◽

S Miura ◽

...

Keyword(s):

Amino Acid ◽

Rat Liver ◽

Coenzyme A ◽

Terminal Region ◽

Proteinase K ◽

Amino Acid Residues ◽

C Terminus ◽

Targeting Signal ◽

Acyl Coenzyme A

To identify the topogenic signal of peroxisomal acyl-coenzyme A oxidase (AOX) of rat liver, we carried out in vitro import experiments with mutant polypeptides of the enzyme. Full-length AOX and polypeptides that were truncated at the N-terminal region were efficiently imported into peroxisomes, as determined by resistance to externally added proteinase K. Polypeptides carrying internal deletions in the C-terminal region exhibited much lower import activities. Polypeptides that were truncated or mutated at the extreme C terminus were totally import negative. When the five amino acid residues at the extreme C terminus were attached to some of the import-negative polypeptides, the import activities were rescued. Moreover, the C-terminal 199 and 70 amino acid residues of AOX directed fusion proteins with two bacterial enzymes to peroxisomes. These results are interpreted to mean that the peroxisome targeting signal of AOX residues at the C terminus and the five or fewer residues at the extreme terminus have an obligatory function in targeting. The C-terminal internal region also has an important role for efficient import, possibly through a conformational effect.

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Importance of C-Terminal Knot Structure in Carbonic Anhydrase II (Part I): Role of C-Terminal Knot Forming GLN253 on Enzymatic Property of Human Carbinic II

Journal of Bio-Science ◽

10.3329/jbs.v14i0.435 ◽

1970 ◽

Vol 14 ◽

pp. 1-9

Author(s):

Mohammad Taufiq Alam

Keyword(s):

Amino Acid ◽

Carbonic Anhydrase ◽

Point Mutation ◽

Amino Acid Residue ◽

Active Site ◽

Terminal Region ◽

Carbonic Anhydrase Ii ◽

C Terminus ◽

Human Carbonic Anhydrase

In both, bovine and human carbonic anhydrase II, a conserved glutamine residue occupies the position in the middle of the knot, which is formed by intercrossing of C-terminal end with N-terminal region. Previous studies have indicated that C-terminus is not the part of an active site, but truncation of 7 amino acid residue in this region can have marked effects on stability of the enzyme (data not published). To gain further insight into the role of specific amino acid residue in C-terminal region, site directed mutagenesis was used to introduce point mutation. Substitution of glutamine with cysteine was chosen because the cysteine residue is less hydrophilic as compared with glutamine and thus, may disrupt the hydrophilic environment in this region. Result indicates that Gln253 located within the C-terminus knot topology plays a significant role in normal function of the enzyme. Thus, C-terminal region might mediate cooperativity between the central active site of the enzyme through proper formation of knot. Key words: Human carbonic anhydrase II; knot topology; point mutation J. bio-sci. 14: 1-9, 2006

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Dual Interactions of the Translational Repressor Paip2 with Poly(A) Binding Protein

Molecular and Cellular Biology ◽

10.1128/mcb.21.15.5200-5213.2001 ◽

2001 ◽

Vol 21 (15) ◽

pp. 5200-5213 ◽

Cited By ~ 101

Author(s):

Kianoush Khaleghpour ◽

Avak Kahvejian ◽

Gregory De Crescenzo ◽

Guylaine Roy ◽

Yuri V. Svitkin ◽

...

Keyword(s):

Binding Protein ◽

Direct Interaction ◽

The Other ◽

Initiation Factor ◽

Rna Recognition Motif ◽

Translational Repressor ◽

C Terminus ◽

Eukaryotic Mrnas ◽

Eukaryotic Initiation Factor 4G ◽

Carboxy Terminal

ABSTRACT The cap structure and the poly(A) tail of eukaryotic mRNAs act synergistically to enhance translation. This effect is mediated by a direct interaction of eukaryotic initiation factor 4G and poly(A) binding protein (PABP), which brings about circularization of the mRNA. Of the two recently identified PABP-interacting proteins, one, Paip1, stimulates translation, and the other, Paip2, which competes with Paip1 for binding to PABP, represses translation. Here we studied the Paip2-PABP interaction. Biacore data and far-Western analysis revealed that Paip2 contains two binding sites for PABP, one encompassing a 16-amino-acid stretch located in the C terminus and a second encompassing a larger central region. PABP also contains two binding regions for Paip2, one located in the RNA recognition motif (RRM) region and the other in the carboxy-terminal region. A two-to-one stoichiometry for binding of Paip2 to PABP with two independentKd s of 0.66 and 74 nM was determined. Thus, our data demonstrate that PABP and Paip2 could form a trimeric complex containing one PABP molecule and two Paip2 molecules. Significantly, only the central Paip2 fragment, which binds with high affinity to the PABP RRM region, inhibits PABP binding to poly(A) RNA and translation.

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Suppression of mutations in two Saccharomyces cerevisiae genes by the adenovirus E1A protein.

Molecular and Cellular Biology ◽

10.1128/mcb.15.6.3227 ◽

1995 ◽

Vol 15 (6) ◽

pp. 3227-3237 ◽

Cited By ~ 22

Author(s):

H A Zieler ◽

M Walberg ◽

P Berg

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Single Gene ◽

Adenovirus E1a ◽

C Terminus ◽

Acid Protein ◽

Conserved Region ◽

Mutant Phenotypes ◽

Yeast Genes ◽

Amino Acid Protein

The protein products of the adenoviral E1A gene are implicated in a variety of transcriptional and cell cycle events, involving interactions with several proteins present in human cells, including parts of the transcriptional machinery and negative regulators of cell division such as the Rb gene product and p107. To determine if there are functional homologs of E1A in Saccharomyces cerevisiae, we have developed a genetic screen for mutants that depend on E1A for growth. The screen is based on a colony color sectoring assay which allows the identification of mutants dependent on the maintenance and expression of an E1A-containing plasmid. Using this screen, we have isolated five mutants that depend on expression of the 12S or 13S cDNA of E1A for growth. A plasmid shuffle assay confirms that the plasmid-dependent phenotype is due to the presence of either the 12S or the 13S E1A cDNA and that both forms of E1A rescue growth of all mutants equally well. The five mutants fall into two classes that were named web1 and web2 (for "wants E1A badly"). Plasmid shuffle assays with mutant forms of E1A show that conserved region 1 (CR1) is required for rescue of the growth of the web1 and web2 E1A-dependent yeast mutants, while the N-terminal 22 amino acids are only partially required; conserved region 2 (CR2) and the C terminus are dispensable. The phenotypes of mutants in both the web1 and the web2 groups are due to a single gene defect, and the yeast genes that fully complement the mutant phenotypes of both groups were cloned. The WEB1 gene sequence encodes a 1,273-amino-acid protein that is identical to SEC31, a protein involved in the budding of transport vesicles from the endoplasmic reticulum. The WEB2 gene encodes a 1,522-amino-acid protein with homology to nucleic acid-dependent ATPases. Deletion of either WEB1 or WEB2 is lethal. Expression of E1A is not able to rescue the lethality of either the web1 or the web2 null allele, implying allele-specific mutations that lead to E1A dependence.

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The PEST-like sequence of I kappa B alpha is responsible for inhibition of DNA binding but not for cytoplasmic retention of c-Rel or RelA homodimers.

Molecular and Cellular Biology ◽

10.1128/mcb.15.2.872 ◽

1995 ◽

Vol 15 (2) ◽

pp. 872-882 ◽

Cited By ~ 77

Author(s):

M K Ernst ◽

L L Dunn ◽

N R Rice

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Direct Interaction ◽

Terminal Region ◽

Nf Kappa B ◽

I Kappa B Alpha ◽

Functional Capabilities ◽

C Terminus

In most cells, proteins belonging to the Rel/NF-kappa B family of transcription factors are held in inactive form in the cytoplasm by an inhibitor protein, I kappa B alpha. Stimulation of the cells leads to degradation of the inhibitor and transit of active DNA-binding Rel/NF-kappa B dimers to the nucleus. I kappa B alpha is also able to inhibit DNA binding by Rel/NF-kappa B dimers in vitro, suggesting that it may perform the same function in cells when the activating signal is no longer present. Structurally, the human I kappa B alpha molecule can be divided into three sections: a 70-amino-acid N terminus with no known function, a 205-residue midsection composed of six ankyrin-like repeats, and a very acidic 42-amino-acid C terminus that resembles a PEST sequence. In this study we examined how the structural elements of the I kappa B alpha protein correlate with its functional capabilities both in vitro and in vivo. Using a battery of I kappa B alpha mutants, we show that (i) a dimer binds a single I kappa B alpha molecule, (ii) the acidic C-terminal region of I kappa B alpha is not required for protein-protein binding and does not mask the nuclear localization signal of the dimer, (iii) the same C-terminal region is required for inhibition of DNA binding, and (iv) this inhibition may be accomplished by direct interaction between the PEST-like region and the DNA-binding region of one of the subunits of the dimer.

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The TATA-Binding Protein (TBP) from the Human Fungal PathogenCandida albicans Can Complement Defects in Human and Yeast TBPs

Journal of Bacteriology ◽

10.1128/jb.180.7.1771-1776.1998 ◽

1998 ◽

Vol 180 (7) ◽

pp. 1771-1776 ◽

Cited By ~ 2

Author(s):

Ping Leng ◽

Philip E. Carter ◽

Alistair J. P. Brown

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Transcription Initiation ◽

Binding Protein ◽

Tata Binding Protein ◽

Binding Domain ◽

Amino Acid Sequences ◽

Initiation Factor ◽

Dna Binding Domain

ABSTRACT Candida albicans is the major fungal pathogen in humans, yet little is known about transcriptional regulation in this organism. Therefore, we have isolated, characterized, and expressed theC. albicans TATA-binding protein (TBP) gene (TBP1), because this general transcription initiation factor plays a key role in the activation and regulation of eukaryotic promoters. Southern and Northern blot analyses suggest that a single C. albicans TBP1 locus is expressed at similar levels in the yeast and hyphal forms of this fungus. The TBP1 open reading frame is 716 bp long and encodes a functional TBP of 27 kDa. C. albicans TBP is capable of binding specifically to a TATA box in vitro, substituting for the human TBP to activate basal transcription in vitro, and suppressing the lethal Δspt15 mutation inSaccharomyces cerevisiae. The predicted amino acid sequences of TBPs from C. albicans and other organisms reveal a striking pattern of C-terminal conservation and N-terminal variability: the C-terminal DNA-binding domain displays at least 80% amino acid sequence identity to TBPs from fungi, flies, nematodes, slime molds, plants, and humans. Sequence differences between human and fungal TPBs in the DNA-binding domain may represent potential targets for antifungal therapy.

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Identification of High-Affinity PB1-Derived Peptides with Enhanced Affinity to the PA Protein of Influenza A Virus Polymerase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01419-10 ◽

2010 ◽

Vol 55 (2) ◽

pp. 696-702 ◽

Cited By ~ 39

Author(s):

Kerstin Wunderlich ◽

Mindaugas Juozapaitis ◽

Charlene Ranadheera ◽

Ulrich Kessler ◽

Arnold Martin ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Binding Affinity ◽

Influenza A Virus ◽

Influenza A ◽

Single Amino Acid ◽

Binding Region ◽

Binding Studies ◽

Relationship Analysis ◽

The Core

ABSTRACTThe influenza A virus polymerase complex, consisting of the subunits PB1, PB2, and PA, represents a promising target for the development of new antiviral drugs. We have previously demonstrated the feasibility of targeting the protein-protein interaction domain between PA and PB1 using peptides derived from the extreme N terminus of PB1 (amino acids [aa] 1 to 15), comprising the PA-binding domain of PB1. To increase the binding affinity of these peptides, we performed a systematic structure-affinity relationship analysis. Alanine and aspartic acid scans revealed that almost all amino acids in the core binding region (aa 5 to 11) are indispensable for PA binding. Using a library of immobilized peptides representing all possible single amino acid substitutions, we were able to identify amino acid positions outside the core PA-binding region (aa 1, 3, 12, 14, and 15) that are variable and can be replaced by affinity-enhancing residues. Surface plasmon resonance binding studies revealed that combination of several affinity-enhancing mutations led to an additive effect. Thus, the feasibility to enhance the PA-binding affinity presents an intriguing possibility to increase antiviral activity of the PB1-derived peptide and one step forward in the development of an antiviral drug against influenza A viruses.

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Eukaryotic Translation Initiation Factor 4E (eIF4E) Binding Site and the Middle One-Third of eIF4GI Constitute the Core Domain for Cap-Dependent Translation, and the C-Terminal One-Third Functions as a Modulatory Region

Molecular and Cellular Biology ◽

10.1128/mcb.20.2.468-477.2000 ◽

2000 ◽

Vol 20 (2) ◽

pp. 468-477 ◽

Cited By ~ 134

Author(s):

Shigenobu Morino ◽

Hiroaki Imataka ◽

Yuri V. Svitkin ◽

Tatyana V. Pestova ◽

Nahum Sonenberg

Keyword(s):

Binding Site ◽

Terminal Region ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Middle Region ◽

Globin Mrna ◽

Ribosome Binding ◽

Amino Terminal ◽

The Core ◽

Core Sequence

ABSTRACT The mammalian eukaryotic initiation factor 4GI (eIF4GI) may be divided into three roughly equal regions; an amino-terminal one-third (amino acids [aa] 1 to 634), which contains the poly(A) binding protein (PABP) and eIF4E binding sites; a middle third (aa 635 to 1039), which binds eIF4A and eIF3; and a carboxy-terminal third (aa 1040 to 1560), which harbors a second eIF4A binding site and a docking sequence for the Ser/Thr kinase Mnk1. Previous reports demonstrated that the middle one-third of eIF4GI is sufficient for cap-independent translation. To delineate the eIF4GI core sequence required for cap-dependent translation, various truncated versions of eIF4GI were examined in an in vitro ribosome binding assay with β-globin mRNA. A sequence of 540 aa encompassing aa 550 to 1090, which contains the eIF4E binding site and the middle region of eIF4GI, is the minimal sequence required for cap-dependent translation. In agreement with this, a point mutation in eIF4GI which abolished eIF4A binding in the middle region completely inhibited ribosomal binding. However, the eIF4GI C-terminal third region, which does not have a counterpart in yeast, modulates the activity of the core sequence. When the eIF4A binding site in the C-terminal region of eIF4GI was mutated, ribosome binding was decreased three- to fourfold. These data indicate that the interaction of eIF4A with the middle region of eIF4GI is necessary for translation, whereas the interaction of eIF4A with the C-terminal region plays a modulatory role.

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