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Cap hydrolysis by Dcp2 is a critical step in several eukaryotic mRNA decay pathways. Processing requires access to cap-proximal nucleotides and the coordinated assembly of a decapping mRNP, but the mechanism of substrate recognition and regulation by protein interactions have remained elusive. Using NMR spectroscopy and kinetic analyses, we show that yeast Dcp2 resolves interactions with the cap and RNA body using a bipartite surface that forms a channel intersecting the catalytic and regulatory Dcp1-binding domains. The interaction with cap is weak but specific and requires binding of the RNA body to a dynamic interface. The catalytic step is stimulated by Dcp1 and its interaction domain, likely through a substrate-induced conformational change. Thus, activation of the decapping mRNP is restricted by access to 5'-proximal nucleotides, a feature that could act as a checkpoint in mRNA metabolism.

Dcp1/2 is the major eukaryotic RNA decapping complex, comprised of the enzyme Dcp2 and activator Dcp1, which removes the 5′ m7G cap from mRNA, committing the transcript to degradation. Dcp1/2 activity is crucial for RNA quality control and turnover, and deregulation of these processes may lead to disease development. The molecular details of Dcp1/2 catalysis remain elusive, in part because both cap substrate (m7GpppN) and m7GDP product are bound by Dcp1/2 with weak (mM) affinity. In order to find inhibitors to use in elucidating the catalytic mechanism of Dcp2, we screened a small library of synthetic m7G nucleotides (cap analogs) bearing modifications in the oligophosphate chain. One of the most potent cap analogs, m7GpSpppSm7G, inhibited Dcp1/2 20 times more efficiently than m7GpppN or m7GDP. NMR experiments revealed that the compound interacts with specific surfaces of both regulatory and catalytic domains of Dcp2 with submillimolar affinities. Kinetics analysis revealed that m7GpSpppSm7G is a mixed inhibitor that competes for the Dcp2 active site with micromolar affinity. m7GpSpppSm7G-capped RNA undergoes rapid decapping, suggesting that the compound may act as a tightly bound cap mimic. Our identification of the first small molecule inhibitor of Dcp2 should be instrumental in future studies aimed at understanding the structural basis of RNA decapping and may provide insight toward the development of novel therapeutically relevant decapping inhibitors.

The HIV-1 protein Rev facilitates the nuclear export of intron-containing viral mRNAs by recognizing a structured RNA site, the Rev-response-element (RRE), contained in an intron. Rev assembles as a homo-oligomer on the RRE using its α-helical arginine-rich-motif (ARM) for RNA recognition. One unique feature of this assembly is the repeated use of the ARM from individual Rev subunits to contact distinct parts of the RRE in different binding modes. How the individual interactions differ and how they contribute toward forming a functional complex is poorly understood. Here we examine the thermodynamics of Rev-ARM peptide binding to two sites, RRE stem IIB, the high-affinity site that nucleates Rev assembly, and stem IA, a potential intermediate site during assembly, using NMR spectroscopy and isothermal titration calorimetry (ITC). NMR data indicate that the Rev-IIB complex forms a stable interface, whereas the Rev-IA interface is highly dynamic. ITC studies show that both interactions are enthalpy-driven, with binding to IIB being 20-30 fold tighter than to IA. Salt-dependent decreases in affinity were similar at both sites and predominantly enthalpic in nature, reflecting the roles of electrostatic interactions with arginines. However, the two interactions display strikingly different partitioning between enthalpy and entropy components, correlating well with the NMR observations. Our results illustrate how the variation in binding modes to different RRE target sites may influence the stability or order of Rev-RRE assembly and disassembly, and consequently its function.

The transcriptional activity of many sequence-specific DNA binding proteins is directly regulated by posttranslational covalent modification. Although this form of regulation was first described nearly two decades ago, it remains poorly understood at a mechanistic level. The prototype for a transcription factor controlled by posttranslational modification is E. coli Ada protein, a chemosensor that both repairs methylation damage in DNA and coordinates the resistance response to genotoxic methylating agents. Ada repairs methyl phosphotriester lesions in DNA by transferring the aberrant methyl group to one of its own cysteine residues; this site-specific methylation enhances tremendously the DNA binding activity of the protein, thereby enabling it to activate a methylation-resistance regulon. Here, we report solution and X-ray structures of the Cys-methylated chemosensor domain of Ada bound to DNA. The structures reveal that both phosphotriester repair and methylation-dependent transcriptional activation function through a zinc- and methylation-dependent electrostatic switch.

Eukaryotic initiation factor 4E (eIF4E) has central roles in the control of several aspects of post-transcriptional gene expression and thereby affects developmental processes. It is also implicated in human diseases. This review explores the relationship between structural, biochemical and biophysical aspects of eIF4E and its function in vivo, including both long-established roles in translation and newly emerging ones in nuclear export and mRNA decay pathways.

The eukaryotic initiation factor 4G (eIF4G) is the core of a multicomponent switch controlling gene expression at the level of translation initiation. It interacts with the small ribosomal subunit interacting protein, eIF3, and the eIF4E/cap-mRNA complex in order to load the ribosome onto mRNA during cap-dependent translation. We describe the solution structure of the complex between yeast eIF4E/cap and eIF4G (393–490). Binding triggers a coupled folding transition of eIF4G (393–490) and the eIF4E N terminus resulting in a molecular bracelet whereby eIF4G (393–490) forms a right-handed helical ring that wraps around the N terminus of eIF4E. Cofolding allosterically enhances association of eIF4E with the cap and is required for maintenance of optimal growth and polysome distributions in vivo. Our data explain how mRNA, eIF4E, and eIF4G exists as a stable mRNP that may facilitate multiple rounds of ribosomal loading during translation initiation, a key determinant in the overall rate of protein synthesis.

A method for measuring intermolecular NOEs in protein complexes based on asymmetric sample deuteration is described. 13C/1H-I,L,V-methyl, U-2H labeled protein is produced using the biosynthetic precursors [γ-13C]-α-ketobutyrate and [γ,γ′-13C2]-α-ketoisovalerate. The labeled protein is mixed with its unlabeled binding partner and a 3D 13C-HMQC-NOESY is recorded, yielding unambiguous intermolecular aromatic/methyl NOEs. A simple synthesis of the biosynthetic precursors via reaction of diethyl oxalate with alkyl Grignard compounds is reported. The method is demonstrated for a 35 kDa heterodimeric protein complex dissolved in a CHAPS micelle. This approach will facilitate the solution structure determination of protein/protein, protein/ligand or protein/nucleic acid complexes.

The increasing diversity of small molecule libraries has been an important source for the development of new drugs and, more recently, for unraveling the mechanisms of cellular events-a process termed chemical genetics.(1) Unfortunately, the majority of currently available compounds are mechanism-based enzyme inhibitors, whereas most of cellular activity regulation proceeds on the level of protein-protein interactions. Hence, the development of small molecule inhibitors of protein-protein interactions is important. When screening compound libraries, low-micromolar inhibitors of protein interactions can be routinely found. The enhancement of affinities and rationalization of the binding mechanism require structural information about the protein-ligand complexes. Crystallization of low-affinity complexes is difficult, and their NMR analysis suffers from exchange broadening, which limits the number of obtainable intermolecular constraints. Here we present a novel method of ligand validation and optimization, which is based on the combination of structural and computational approaches. We successfully used this method to analyze the basis for structure-activity relationships of previously selected (2) small molecule inhibitors of the antiapoptotic protein Bcl-xL and identified new members of this inhibitor family.

Like many viruses, HIV hijacks the host cell's apparatus for normal protein ubiquitination and degradation, using it to eliminate antiviral proteins. Understanding how a virus recruits and targets the ubqiuitination complex is critical for developing therapeutics to prevent it. One HIV protein responsible for this hijacking is Virion infectivity factor (Vif). Vif is intrinsically disordered but loses flexibility as it binds more host proteins, a process that may be crucial for function. We are investigating the complex formed with Vif and the host proteins EloB, EloC, and CBF-beta. Using molecular dynamics simulations and NMR spectroscopy we have found that this Vif-host protein complex exhibits global dynamics and occupies alternate conformational states. We have also characterized how these motions are affected by the binding of an additional host protein, Cul5. Understanding the dynamics and conformational ensemble of the Vif-host protein complex could reveal future directions for therapeutics.

Uveitis is not infrequently reported as an ophthalmic manifestation of multiple sclerosis. This is usually in the form of a peripheral uveitis, but granulomatous uveitis has occasionally also been described. The patient in this case study was hospitalized for signs and symptoms suggestive of multiple sclerosis, which was later confirmed by neurologic exam and magnetic resonance imaging. In addition to internuclear ophthalmoplegia causing complaints of diplopia, a granulomatous uveitis in the right eye was found, contributing to a decreased acuity in that eye. Other neurological inflammatory causes were ruled out, and the uveitis was attributed to the multiple sclerosis.

We describe new solid-state NMR methods for measuring the magnitude of the chemical shift anisotropy (CSA) and/or the heteronuclear dipolar coupling in the presence of homonuclear dipolar coupled spin networks under magic-angle spinning (MAS). The techniques employ 2π/5 phase shifts of a frequency switched Lee–Goldburg irradiation scheme which recouples the CSA and/or the heteronuclear dipolar coupling while simultaneously decoupling the homonuclear dipolar interactions. The induced spin dynamics are sensitive to the magnitude of the CSA and its asymmetry and may be implemented under conditions where the MAS rate exceeds the size of the anisotropic interactions. These approaches could find use in measurements of the magnitudes and relative orientations of CSA and/or heteronuclear dipolar couplings to extract torsion angles in uniformly labeled systems.

A technique for amplifying the apparent magnitudes of 13 C‐ 1 H v r /2 pud d/ 3, where dd is the dipolar coupling strength, the and 15 N‐ 1 H dipolar interactions in magic-angle spinning experi- dipolar spectrum is dominated by an uninformative cenments is described. By inserting rotor-synchronized 1807 pulses in terband. For a one-bond N ‐ H dipolar coupling measured the evolution period of a 2D dipolar-chemical shift experiment, with MREV-8 homonuclear decoupling, dd E 4.5 kHz and heteronuclear dipolar couplings are doubled or quadrupled rela- the centerband dominates for spinning speeds as low as vr / tive to the spinning speed. The increased number of dipolar side- 2pE 1.5 kHz. While low spinning speeds are desirable for bands is desirable for retaining structural information in the indi- extracting structural information, the necessity of resolving rectly detected dipolar dimension while resolving inequivalent sites different chemical moieties through isotropic chemical shifts in the isotropic chemical shift dimension at relatively high spinresults in an opposing constraint. Higher spinning speeds ning speeds. This coupling amplification method is incorporated into an experiment that determines the peptide torsion angle f average the chemical shift anisotropy (dCSA) more comthrough the relative orientation of the C a ‐H a and N‐H N bonds. pletely and thus yield better-resolved spectra. This conflict It is shown both experimentally and theoretically that the angular is exacerbated at higher magnetic field strengths, since shift resolution of the measurement is enhanced significantly by the anisotropies scale linearly with the field strength while the selective doubling of the N‐H N coupling. q 1997 Academic Press dipolar couplings are field-independent. Thus, the desirable

A solid-state NMR experiment is presented for measuring Ψ torsion angles in (15N–13Cα–13CO–15N)-labeled peptides. Creation of 13Cα–13CO double-quantum coherence (DQC) is followed by selective evolution under 15N–13C dipolar interactions. The time-course of 13Cα–13CO DQC dephasing is sensitive to the Ψ torsion angle, particularly for values associated with β-sheet secondary structure (120–180°). We apply the technique to glycylglycine⋅HCl and extract a value for Ψ that matches the diffraction-determined value (Ψ=162.1°C) with a precision of ±5°.

We recently reported that two homologous yeast proteins, Rai1 and Dxo1, function in a quality control mechanism to clear cells of incompletely 5′-end capped mRNAs. Here we report that their mammalian homolog, Dom3Z, possesses pyrophosphohydrolase, decapping and 5′-3′ exoribonuclease activities, and will be referred to as DXO. Surprisingly, we find that DXO preferentially degrades defectively capped pre-mRNAs in cells. Further studies show that incompletely capped pre-mRNAs are inefficiently spliced at all introns, in contrast to current understanding, and poorly cleaved for polyadenylation. Crystal structures of DXO in complex with substrate mimic and products at up to 1.5Å resolution provide elegant insights into the catalytic mechanism and molecular basis for its three apparently distinct activities. Our data reveal a pre-mRNA 5′-end capping quality control mechanism in mammalian cells, with DXO as the central player for this mechanism, and demonstrate an unexpected intimate link between proper 5′-end capping and subsequent pre-mRNA processing.

The HIV-1 accessory protein Vif hijacks a cellular Cullin-RING ubiquitin ligase, CRL5, to promote degradation of the APOBEC3 (A3) family of restriction factors. Recently, the cellular transcription cofactor CBFβ was shown to form a complex with CRL5-Vif and to be essential for A3 degradation and viral infectivity. We now demonstrate that CBFβ is required for assembling a well-ordered CRL5-Vif complex by inhibiting Vif oligomerization and by activating CRL5-Vif via direct interaction. The CRL5-Vif-CBFβ holoenzyme forms a well-defined heterohexamer, indicating that Vif simultaneously hijacks CRL5 and CBFβ. Heterodimers of CBFβ and RUNX transcription factors contribute toward the regulation of genes, including those with immune system functions. We show that binding of Vif to CBFβ is mutually exclusive with RUNX heterodimerization and impacts the expression of genes whose regulatory domains are associated with RUNX1. Our results provide a mechanism by which a pathogen with limited coding capacity uses one factor to hijack multiple host pathways.

There is a wide gap between the generation of large-scale biological data sets and more-detailed, structural and mechanistic studies. However, recent studies that explicitly combine data from systems and structural biological approaches are having a profound effect on our ability to predict how mutations and small molecules affect atomic-level mechanisms, disrupt systems-level networks, and ultimately lead to changes in organismal fitness. In fact, we argue that a shared framework for analysis of nonadditive genetic and thermodynamic responses to perturbations will accelerate the integration of reductionist and global approaches. A stronger bridge between these two areas will allow for a deeper and more-complete understanding of complex biological phenomenon and ultimately provide needed breakthroughs in biomedical research.

International audience

Glucocorticoid receptor binds to genomic response elements and regulates gene transcription with cell- and gene-specificity. Within a response element, the precise sequence to which the receptor binds has been implicated in directing its structure and activity. We use NMR chemical shift difference mapping to show that non-specific interactions with particular base positions within the binding sequence, such as those of the “spacer”, affect the conformation of distinct regions of the rat glucocorticoid receptor DNA binding domain. These regions include the DNA-binding surface, the “lever arm” and the dimerization interface, suggesting an allosteric pathway that signals between the DNA binding sequence and the associated dimer partner. Disrupting this path by mutating the dimer interface alters sequence-specific conformations, DNA-binding kinetics and transcriptional activity. Our study demonstrates that glucocorticoid receptor dimer partners collaborate to read DNA shape and to direct sequence specific gene activity.

Restriction factors, such as the retroviral complementary DNA deaminase APOBEC3G, are cellular proteins that dominantly block virus replication1-3. The AIDS virus, human immunodeficiency virus type 1 (HIV-1), produces the accessory factor Vif, which counteracts the host’s antiviral defence by hijacking a ubiquitin ligase complex, containing CUL5, ELOC, ELOB and a RING-box protein, and targeting APOBEC3G for degradation4-10. Here we reveal, using an affinity tag/purification mass spectrometry approach, that Vif additionally recruits the transcription cofactor CBF-β to this ubiquitin ligase complex. CBF-β, which normally functions in concert with RUNX DNA binding proteins, allows the reconstitution of a recombinant six-protein assembly that elicits specific polyubiquitination activity with APOBEC3G, but not the related deaminase APOBEC3A. Using RNA knockdown and genetic complementation studies, we also demonstrate that CBF-β is required for Vif-mediated degradation of APOBEC3G and therefore for preserving HIV-1 infectivity. Finally, simian immunodeficiency virus (SIV) Vif also binds to and requires CBF-β to degrade rhesus macaque APOBEC3G, indicating functional conservation. Methods of disrupting the CBF-β–Vif interaction might enable HIV-1 restriction and provide a supplement to current antiviral therapies that primarily target viral proteins.

Decapping by Dcp2 is an essential step in 5'-to-3' mRNA decay. In yeast, decapping requires an open-to-closed transition in Dcp2, though the link between closure and catalysis remains elusive. Here we show using NMR that cap binds conserved residues on both the catalytic and regulatory domains of Dcp2. Lesions in the cap-binding site on the regulatory domain reduce the catalytic step by two orders of magnitude and block the formation of the closed state, whereas Dcp1 enhances the catalytic step by a factor of 10 and promotes closure. We conclude that closure occurs during the rate-limiting catalytic step of decapping, juxtaposing the cap-binding region of each domain to form a composite active site. This work suggests a model for regulation of decapping where coactivators trigger decapping by stabilizing a labile composite active site.

Escherichia coli spheroplast protein y (EcSpy) is a small periplasmic protein that is homologous with CpxP, an inhibitor of the extracytoplasmic stress response. Stress conditions such as spheroplast formation induce the expression of Spy via the Cpx or the Bae two-component systems in E. coli, though the function of Spy is unknown. Here, we report the crystal structure of EcSpy, which reveals a long kinked hairpin-like structure of four α-helices that form an antiparallel dimer. The dimer contains a curved oval shape with a highly positively charged concave surface that may function as a ligand binding site. Sequence analysis reveals that Spy is highly conserved over the Enterobacteriaceae family. Notably, three conserved regions that contain identical residues and two LTxxQ motifs are placed at the horizontal end of the dimer structure, stabilizing the overall fold. CpxP also contains the conserved sequence motifs and has a predicted secondary structure similar to Spy, suggesting that Spy and CpxP likely share the same fold.

Cap hydrolysis is a critical step in several eukaryotic mRNA decay pathways and is carried out by the evolutionarily conserved decapping complex containing Dcp2 at the catalytic core. In yeast, Dcp1 is an essential activator of decapping and coactivators such as Edc1 and Edc2 are thought to enhance activity, though their mechanism remains elusive. Using kinetic analysis we show that a crucial function of Dcp1 is to couple the binding of coactivators of decapping to activation of Dcp2. Edc1 and Edc2 bind Dcp1 via its EVH1 proline recognition site and stimulate decapping by 1000-fold, affecting both the KM for mRNA and rate of the catalytic step. The C-terminus of Edc1 is necessary and sufficient to enhance the catalytic step, while the remainder of the protein likely increases mRNA binding to the decapping complex. Lesions in the Dcp1 EVH1 domain or the Edc1 proline-rich sequence are sufficient to block stimulation. These results identify a new role of Dcp1, which is to link the binding of coactivators to substrate recognition and activation of Dcp2.

The stability of all RNA polymerase II transcripts depends on the 5'-terminal cap structure. Removal of the cap is a prerequisite for 5' to 3'-decay and is catalyzed by distinct cellular and viral decapping activities. Over the past decade, several decapping enzymes have been characterized through functional and structural studies. An emerging theme is that function is regulated by protein interactions; however, in vitro assays to dissect the effects on enzyme activity are unavailable. Here we present a kinetic assay to monitor decapping by the heterodimeric yeast Dcp1/Dcp2 complex. Kinetic constants related to RNA binding and the rate of the catalytic step can be determined with recombinant enzyme and cap-radiolabeled RNA substrate, allowing substrate specificity and the role of activating factors to be firmly established.

Conformational dynamics are thought to play an important role in ligand binding and catalysis by cytochrome P450 enzymes, but few techniques exist to examine them in molecular detail. Using a unique isotopic labeling strategy, we have site specifically inserted a (13)C-labeled unnatural amino acid residue, (13)C-p-methoxyphenylalanine (MeOF), into two different locations in the substrate binding region of the thermophilic cytochrome P450 enzyme CYP119. Surprisingly, in both cases the resonance signal from the ligand-free protein is represented by a doublet in the (1)H,(13)C-HSQC spectrum. Upon binding of 4-phenylimidazole, the signals from the initial resonances are reduced in favor of a single new resonance, in the case of the F162MeOF mutant, or two new resonances, in the case of the F153MeOF mutant. This represents the first direct physical evidence for the ligand-dependent existence of multiple P450 conformers simultaneously in solution. This general approach may be used to further illuminate the role that conformational dynamics plays in the complex enzymatic phenomena exhibited by P450 enzymes.

SUMO modification of nuclear receptors, including the constitutively active receptor steroidogenic factor 1 (SF-1; NR5A1), is proposed to repress their transcriptional activity. We examined the functional and structural consequences of SF-1 sumoylation at two conserved lysines (Lys119 and Lys194) that reside adjacent to the DNA-binding domain (DBD) and ligand-binding domain (LBD), respectively. Surprisingly, while previous loss-of-function studies predicted that sumoylation at Lys194 would greatly impact SF-1 function, the conformation and coregulator recruitment of fully sumoylated SF-1 LBD protein was either unchanged or modestly impaired. Sumoylation at Lys194 also modestly reduced Ser203 phosphorylation. In contrast to these findings, sumoylation of the DBD at Lys119 resulted in a marked and selective loss of DNA binding to noncanonical SF-1 targets, such as inhibinalpha; this binding deficit was extended to all sites when the sumoylated human mutant (R92Q) protein, which exhibits lower activity, was used. Consistent with this result, the K119R mutant, compared to wild-type SF-1, was selectively recruited to a "SUMO-sensitive" site in the endogenous inhibinalpha promoter, leading to increased transcription. DNA binding and sumoylation of Lys119 appeared to be mutually exclusive, suggesting that once SF-1 is bound to DNA, sumoylation may be less important in regulating SF-1 activity. We propose that sumoylation of nuclear receptors imposes an active posttranslational mark that dampens recognition of SUMO-sensitive target genes to restrain their expression.

The stability of all RNA polymerase II transcripts depends on the 5′-terminal cap structure. Removal of the cap is a prerequisite for 5′ to 3′-decay and is catalyzed by distinct cellular and viral decapping activities. Over the past decade, several decapping enzymes have been characterized through functional and structural studies. An emerging theme is that function is regulated by protein interactions; however, in vitro assays to dissect the effects on enzyme activity are unavailable. Here we present a kinetic assay to monitor decapping by the heterodimeric yeast Dcp1/Dcp2 complex. Kinetic constants related to RNA binding and the rate of the catalytic step can be determined with recombinant enzyme and cap-radiolabeled RNA substrate, allowing substrate specificity and the role of activating factors to be firmly established.

A comprehensive structural analysis of the complete and unmodified Sup35 prion domain in two distinct infectious conformations is presented. A variety of techniques is used to provide structural details of these two prion conformations, one weakly and one strongly propagating strain. The data show that the fibril conformation of both strains share a common amyloid-like core, comprising the glutamine/asparagine rich first 40 residues. In the weaker strain this stable structure is dramatically expanded to 70 amino acids. Among the many surprises to arise from studies of prion biology, perhaps the most unexpected is the strain phenomenon whereby a single protein can misfold into structurally distinct, infectious states that cause distinguishable phenotypes1,2,3. Similarly, proteins can adopt a spectrum of conformations in non-infectious diseases of protein folding; some are toxic and others are well tolerated4. However, our understanding of the structural differences underlying prion strains and how these differences alter their physiological impact remains limited. Here we use a combination of solution NMR, amide hydrogen/deuterium (H/D) exchange and mutagenesis to study the structural differences between two strain conformations, termed Sc4 and Sc37 (ref. 5), of the yeast Sup35 prion. We find that these two strains have an overlapping amyloid core spanning most of the Gln/Asn-rich first 40 amino acids that is highly protected from H/D exchange and very sensitive to mutation. These features indicate that the cores are composed of tightly packed β-sheets possibly resembling ‘steric zipper’ structures revealed by X-ray crystallography of Sup35-derived peptides6,7. The stable structure is greatly expanded in the Sc37 conformation to encompass the first 70 amino acids, revealing why this strain shows increased fibre stability and decreased ability to undergo chaperone-mediated replication8. Our findings establish that prion strains involve large-scale conformational differences and provide a structural basis for understanding a broad range of functional studies, including how conformational changes alter the physiological impact of prion strains.

Eukaryotic mRNA decapping by Dcp2 is the penultimate step in several mRNA decay pathways. To understand regulation of Dcp2 by ligand interactions, we have assigned the backbone and sidechain methyl Ile (delta1), Leu and Val chemical shifts of the catalytic domain of the S. Cerevisiae enzyme.

International audience; Assembly of the eIF4E/eIF4G complex has a central role in the regulation of gene expression at the level of translation initiation. This complex is regulated by the 4E-BPs, which compete with eIF4G for binding to eIF4E and which have tumor-suppressor activity. To pharmacologically mimic 4E-BP function we developed a high-throughput screening assay for identifying small-molecule inhibitors of the eIF4E/eIF4G interaction. The most potent compound identified, 4EGI-1, binds eIF4E, disrupts eIF4E/eIF4G association, and inhibits cap-dependent translation but not initiation factor-independent translation. While 4EGI-1 displaces eIF4G from eIF4E, it effectively enhances 4E-BP1 association both in vitro and in cells. 4EGI-1 inhibits cellular expression of oncogenic proteins encoded by weak mRNAs, exhibits activity against multiple cancer cell lines, and appears to have a preferential effect on transformed versus nontransformed cells. The identification of this compound provides a new tool for studying translational control and establishes a possible new strategy for cancer therapy.

Protein structures up to 25 kDa may be obtained using distance and torsion angle restraints provided by the nuclear Overhauser effect (NOE) and J couplings respectively. Alternatively, distances may be obtained between a paramagnetic center and a nucleus through the electron-nuclear dipolar interaction. A paramagnetic center may be introduced into an intrinsically diamagnetic protein by covalently attaching a nitroxide spin label to cysteine. This technique, referred to as site-directed spin labeling (SDSL), has been employed extensively in electron-paramagnetic resonance. We review applications of SDSL to obtain global folds of proteins and macromolecular complexes within the frame work of high-resolution NMR. Issues pertaining to protein engineering, interpretation of paramagnetic broadening and combination of paramagnetic broadening with limited NOE data will be discussed. Paramagnetic broadening effects (PBEs) in conjunction with limited NOE data and/or residual dipolar couplings and TROSY triple resonance methodology promise to extend the size limit of proteins amenable to structural studies by NMR

The cap-binding protein eIF4E is the first in a chain of translation initiation factors that recruit 40S ribosomal subunits to the 5' end of eukaryotic mRNA. During cap-dependent translation, this protein binds to the 5'-terminal m(7)Gppp cap of the mRNA, as well as to the adaptor protein eIF4G. The latter then interacts with small ribosomal subunit-bound proteins, thereby promoting the mRNA recruitment process. Here, we show apo-eIF4E to be a protein that contains extensive unstructured regions, which are induced to fold upon recognition of the cap structure. Binding of eIF4G to apo-eIF4E likewise induces folding of the protein into a state that is similar to, but not identical with, that of cap-bound eIF4E. At the same time, binding of each of the binding partners of eIF4E modulates the kinetics with which it interacts with the other partner. We present structural, kinetic and mutagenesis data that allow us to deduce some of the detailed folding transitions that take place during the eIF4E interactions.

Soluble methane monooxygenase (sMMO) catalyzes the hydroxylation of methane by dioxygen to methanol, the first step in carbon assimilation by methanotrophs. This multicomponent system transfers electrons from NADH through a reductase component to the non-heme diiron center in the hydroxylase where O(2) is activated. The reductase component comprises three distinct domains, a [2Fe-2S] ferredoxin domain along with FAD- and NADH-binding domains. We report the solution structure of the reduced 27.6 kDa FAD- and NADH-binding domains (MMOR-FAD) of the reductase from Methylococcus capsulatus (Bath). The FAD-binding domain consists of a six-stranded antiparallel beta-barrel and one alpha-helix, with the first 10 N-terminal residues unstructured. In the interface between the two domains, the FAD cofactor is tightly bound in an unprecedented extended conformation. The NADH-binding domain consists of a five-stranded parallel beta-sheet with four alpha-helices packing closely around this sheet. MMOR-FAD is structurally homologous to other FAD-containing oxidoreductases, and we expect similar structures for the FAD/NADH-binding domains of reductases that occur in other multicomponent monooxygenases.

Experiments which require mixing among spins with large frequency differences are generally performed with sequences based on composite pulses or computer-optimized cycles. Adiabatic pulses generally offer several advantages over other approaches, including greater single spin inversion bandwidths and tolerance to RF inhomogeneity. Here, a novel theoretical framework is presented in order to understand how spin–spin interactions are influenced by adiabatic inversion pulses, and insights from this approach are used to design more efficient adiabatic coherence exchange experiments. For very large frequency differences, this new approach generally offers improved results over previously applied mixing sequences, as applied to 13C–13C experiments which are the basis of modern sidechain assignment techniques in proteins. It is also anticipated that the approach presented here will be applicable to the analysis of various alternative approaches to adiabatic mixing.

A method for measuring intermolecular NOEs in protein complexes based on asymmetric sample deuteration is described. (13)C/(1)H-I,L,V-methyl, U-(2)H labeled protein is produced using the biosynthetic precursors [gamma-(13)C]-alpha-ketobutyrate and [gamma,gamma'-(13)C(2)]-alpha-ketoisovalerate. The labeled protein is mixed with its unlabeled binding partner and a 3D (13)C-HMQC-NOESY is recorded, yielding unambiguous intermolecular aromatic/methyl NOEs. A simple synthesis of the biosynthetic precursors via reaction of diethyl oxalate with alkyl Grignard compounds is reported. The method is demonstrated for a 35 kDa heterodimeric protein complex dissolved in a CHAPS micelle. This approach will facilitate the solution structure determination of protein/protein, protein/ligand or protein/nucleic acid complexes.

We demonstrate a dipolar-chemical shift correlation technique for sign-sensitive determination of the torsion angle phi in solid peptides and proteins under magic-angle spinning. The indirect dimension of the experiment is obtained by separate but synchronous evolution of the magnetization under the 15N chemical shift and the C-H dipolar coupling. The resulting sum and difference spectrum of the two frequencies, with more than ten independent sidebands, depends strongly on the relative orientation of the 15N chemical shift tensor and the Calpha-Halpha bond. This relative orientation reflects the C(O)i-1-N-Calpha-C(O)i torsion angle. The technique can distinguish phi angles over the full range of 360 degrees when the amide 15N chemical shift tensor does not possess reflection symmetry with respect to the peptide plane. Thus it complements our previous HNCH experiment, in which two mirror-symmetric conformers of the HN-N bond relative to the Calpha-Halpha bond around the N-Calpha axis cannot be distinguished.

Depuis une quinzaine d'annees, des cas d'interference entre la dynamique de molecules et la manipulation coherente de l'aimantation nucleaire realisee dans le cadre d'experiences de resonance magnetique nucleaire (RMN) ont ete identifies et etudies en detail, qu'il s'agisse du decouplage de spins, de la polarisation croisee ou de la rotation a l'angle magique (magic-angle spinning, ou MAS). Des experiences recentes realisees dans notre laboratoire nous apportent des details concernant la nature d'une perturbation responsable de l'elargissement des raies 13 C et 15 N dans le cas de molecules modeles, celle de l'interference entre un mouvement moleculaire et le decouplage des protons. Le meme effet est demontre ici pour la premiere fois dans le cas d'un peptide membranaire, la gramicidine A (gA), dans une bicouche lipidique hydratee. Les experiences presentees ici constituent la premiere observation d'un spectre RMN solide 13 C a haute resolution d'un C a de peptide membranaire en bicouche lipidique. De plus, le developpement de strategies destinees a contourner l'effet d'elargissement des raies nous permet d'extraire des informations concernant la dynamique de gA dans des conditions physiologiques.

Novel two-dimensional magic-angle spinning (2D MAS) NMR experiments designed to measure the magnitudes and signs of 13C−1H dipolar interactions in fluid phase lipid bilayers are presented. MAS is e...

Cellular restriction factors help to defend humans against human immunodeficiency virus (HIV). HIV accessory proteins hijack at least three different Cullin-RING ubiquitin ligases, which must be activated by the small ubiquitin-like protein NEDD8, in order to counteract host cellular restriction factors. We found that conjugation of NEDD8 to Cullin-5 by the NEDD8-conjugating enzyme UBE2F is required for HIV Vif-mediated degradation of the host restriction factor APOBEC3G (A3G). Pharmacological inhibition of the NEDD8 E1 by MLN4924 or knockdown of either UBE2F or its RING-protein binding partner RBX2 bypasses the effect of Vif, restoring the restriction of HIV by A3G. NMR mapping and mutational analyses define specificity determinants of the UBE2F NEDD8 cascade. These studies demonstrate that disrupting host NEDD8 cascades presents a novel antiretroviral therapeutic approach enhancing the ability of the immune system to combat HIV., Author Summary The APOBEC3 family of editing enzymes catalyzes lethal hypermutation of retroviral genomes to block spread of virus in host. HIV Vif targets APOBEC3 family members for destruction by a cellular ubiquitin ligase containing CUL5. A major goal in the design of the next generation of antiretroviral therapies is to find an inhibitor of Vif so that the activity of the APOBEC3 family of antiretroviral enzymes can be restored. We define a three-enzyme cascade that is required to activate Vif by addition of the ubiquitin-like NEDD8 protein to CUL5. MLN4924, an anti-cancer compound currently in phase 1 clinical trials, inhibits the NEDD8 cascade, blocks the action of Vif, and thus has potent anti-HIV activity. Furthermore, our studies define downstream drug targets in the NEDD8 cascade more selective for inhibition of HIV Vif. We demonstrate pharmacological inhibition of HIV replication through a mechanism that restores the innate immunity provided by APOBEC3 enzymes by targeting a host pathway, providing additional candidates that could be further exploited for therapeutic development. Inhibition of this NEDD8 cascade alone, or in combination with existing antiretroviral drugs could prove to be a useful treatment for HIV.

SummaryHIV-1 encodes the accessory protein Vif, which hijacks a host Cullin-RING ubiquitin ligase (CRL) complex as well as the non-canonical cofactor CBFβ, to antagonize APOBEC3 antiviral proteins. Non-canonical cofactor recruitment to CRL complexes by viral factors, to date, has only been attributed to HIV-1 Vif. To further study this phenomenon, we employed a comparative approach combining proteomic, biochemical, structural, and virological techniques to investigate Vif complexes across the lentivirus genus, including primate (HIV-1 and simian immunodeficiency virus macaque [SIVmac]) and non-primate (FIV, BIV, and MVV) viruses. We find that CBFβ is completely dispensable for the activity of non-primate lentiviral Vif proteins. Furthermore, we find that BIV Vif requires no cofactor and that MVV Vif requires a novel cofactor, cyclophilin A (CYPA), for stable CRL complex formation and anti-APOBEC3 activity. We propose modular conservation of Vif complexes allows for potential exaptation of functions through the acquisition of non-CRL-associated host cofactors while preserving anti-APOBEC3 activity.