Filipp Frank

Structural basis for 5'-nucleotide base-specific recognition of guide RNA by human AGO2.

MicroRNAs (miRNAs) mediate post-transcriptional gene regulation through association with Argonaute proteins (AGOs). Crystal structures of archaeal and bacterial homologues of AGOs have shown that the MID (middle) domain mediates the interaction with the phosphorylated 5′ end of the miRNA guide strand and this interaction is thought to be independent of the identity of the 5′ nucleotide in these systems. However, analysis of the known sequences of eukaryotic miRNAs and co-immunoprecipitation experiments indicate that there is a clear bias for U or A at the 5′ position. Here we report the crystal structure of a MID domain from a eukaryotic AGO protein, human AGO2. The structure, in complex with nucleoside monophosphates (AMP, CMP, GMP, and UMP) mimicking the 5′ end of miRNAs, shows that there are specific contacts made between the base of UMP or AMP and a rigid loop in the MID domain. Notably, the structure of the loop discriminates against CMP and GMP and dissociation constants calculated from NMR titration experiments confirm these results, showing that AMP (0.26 mM) and UMP (0.12 mM) bind with up to 30-fold higher affinity than either CMP (3.6 mM) or GMP (3.3 mM). This study provides structural evidence for nucleotide-specific interactions in the MID domain of eukaryotic AGO proteins and explains the observed preference for U or A at the 5′ end of miRNAs.

miRNA-mediated deadenylation is orchestrated by GW182 through two conserved motifs that interact with CCR4–NOT

miRNAs recruit the miRNA-induced silencing complex (miRISC), which includes Argonaute and GW182 as core proteins. GW182 proteins effect translational repression and deadenylation of target mRNAs. However, the molecular mechanisms of GW182-mediated repression remain obscure. We show here that human GW182 independently interacts with the PAN2–PAN3 and CCR4–NOT deadenylase complexes. Interaction of GW182 with CCR4–NOT is mediated by two newly discovered phylogenetically conserved motifs. Although either motif is sufficient to bind CCR4–NOT, only one of them can promote processive deadenylation of target mRNAs. Thus, GW182 serves as both a platform that recruits deadenylases and as a deadenylase coactivator that facilitates the removal of the poly(A) tail by CCR4–NOT.

A conserved protonation-dependent switch controls drug binding in the Abl kinase

In many protein kinases, a characteristic conformational change (the “DFG flip”) connects catalytically active and inactive conformations. Many kinase inhibitors—including the cancer drug imatinib—selectively target a specific DFG conformation, but the function and mechanism of the flip remain unclear. Using long molecular dynamics simulations of the Abl kinase, we visualized the DFG flip in atomic-level detail and formulated an energetic model predicting that protonation of the DFG aspartate controls the flip. Consistent with our models predictions, we demonstrated experimentally that the kinetics of imatinib binding to Abl kinase have a pH dependence that disappears when the DFG aspartate is mutated. Our model suggests a possible explanation for the high degree of conservation of the DFG motif: that the flip, modulated by electrostatic changes inherent to the catalytic cycle, allows the kinase to access flexible conformations facilitating nucleotide binding and release.

Structural basis for the recruitment of the human CCR4-NOT deadenylase complex by tristetraprolin

Tristetraprolin (TTP) is an RNA-binding protein that controls the inflammatory response by limiting the expression of several proinflammatory cytokines. TTP post-transcriptionally represses gene expression by interacting with AU-rich elements (AREs) in 3′ untranslated regions of target mRNAs and subsequently engenders their deadenylation and decay. TTP accomplishes these tasks, at least in part, by recruiting the multisubunit CCR4–NOT deadenylase complex to the mRNA. Here we identify an evolutionarily conserved C-terminal motif in human TTP that directly binds a central domain of CNOT1, a core subunit of the CCR4–NOT complex. A high-resolution crystal structure of the TTP–CNOT1 complex was determined, providing the first structural insight, to our knowledge, into an ARE-binding protein bound to the CCR4–NOT complex. Mutations at the CNOT1-TTP interface impair TTP-mediated deadenylation, demonstrating the significance of this interaction in TTP-mediated gene silencing.

Arabidopsis Argonaute MID domains use their nucleotide specificity loop to sort small RNAs

The 5′‐nucleotide of small RNAs associates directly with the MID domain of Argonaute (AGO) proteins. In humans, the identity of the 5′‐base is sensed by the MID domain nucleotide specificity loop and regulates the integrity of miRNAs. In Arabidopsis thaliana, the 5′‐nucleotide also controls sorting of small RNAs into the appropriate member of the AGO family; however, the structural basis for this mechanism is unknown. Here, we present crystal structures of the MID domain from three Arabidopsis AGOs, AtAGO1, AtAGO2 and AtAGO5, and characterize their interactions with nucleoside monophosphates (NMPs). In AtAGOs, the nucleotide specificity loop also senses the identity of the 5′‐nucleotide but uses more diverse modes of recognition owing to the greater complexity of small RNAs found in plants. Binding analyses of these interactions reveal a strong correlation between their affinities and evolutionary conservation.

Structural analysis of 5'-mRNA-cap interactions with the human AGO2 MID domain.

In RNA silencing, microRNA (miRNA)‐mediated translational repression occurs through mechanisms that do not invoke messenger‐RNA (mRNA) target cleavage by Argonaute proteins. The nature of these mechanisms is unclear, but several recent studies have proposed that a direct interaction between the mRNA–cap and the middle (MID) domain of Argonautes is involved. Here, we present crystallographic and NMR data demonstrating that cap analogues do not bind significantly to the isolated MID domain of human Argonaute 2 (hAGO2) and are found in the miRNA 5′‐nucleotide binding site in an implausible binding mode. Additionally, in vitro pull‐down experiments with full‐length hAGO2 indicate that the interaction with cap analogues is nonspecific.

An Immunogenic Peptide in the A-box of HMGB1 Protein Reverses Apoptosis-induced Tolerance through RAGE Receptor

Background: The role of caspase-1 in regulating the immunogenic properties of HMGB1 has not been previously reported. Results: We have mapped a peptide in the A-box of HMGB1 that reverses tolerance through RAGE. Conclusion: Inflammasome signaling regulates the immunogenic activity of HMGB1. Significance: Immunogenic peptides within the HMGB1 A-box may be exploited to reverse immune tolerance in sepsis patients. Apoptotic cells trigger immune tolerance in engulfing phagocytes. This poorly understood process is believed to contribute to the severe immunosuppression and increased susceptibility to nosocomial infections observed in critically ill sepsis patients. Extracellular high mobility group box 1 (HMGB1) is an important mediator of both sepsis lethality and the induction of immune tolerance by apoptotic cells. We have found that HMGB1 is sensitive to processing by caspase-1, resulting in the production of a fragment within its N-terminal DNA-binding domain (the A-box) that signals through the receptor for advanced glycation end products (RAGE) to reverse apoptosis-induced tolerance. In a two-hit mouse model of sepsis, we show that tolerance to a secondary infection and its associated mortality were effectively reversed by active immunization with dendritic cells treated with HMGB1 or the A-box fragment, but not a noncleavable form of HMGB1. These findings represent a novel link between caspase-1 and HMGB1, with potential therapeutic implications in infectious and inflammatory diseases.

Molecular dynamics study of chemically engineered green fluorescent protein mutants: Comparison of intramolecular fluorescence resonance energy transfer rate

Because of its unusual spectroscopic properties, green fluorescent protein (GFP) has become a useful tool in molecular genetics, biochemistry and cell biology. Here, we computationally characterize the behavior of two GFP constructs, designed as bioprobes for enzymatic triggering using intramolecular fluorescence resonance energy transfer (FRET). These constructs differ in the location of an intramolecular FRET partner, an attached chemical chromophore (either near an N‐terminal or C‐terminal site). We apply the temperature replica exchange molecular dynamics method to the two flexible constructs in conjunction with a generalized Born implicit solvent model. The calculated rate of FRET was derived from the interchromophore distance, R, and orientational factor, κ2. In agreement with experiment, the construct with the C‐terminally attached dye was predicted to have higher energy transfer rate than observed for the N‐terminal construct. The molecular basis for this observation is discussed. In addition, we find that the orientational factor, κ2, deviates from the commonly assumed value, the implications of which are also considered. Proteins 2009.

Repair of Isoaspartate Formation Modulates the Interaction of Deamidated 4E-BP2 with mTORC1 in Brain

In eukaryotes, a rate-limiting step of translation initiation is recognition of the mRNA 5′ m7GpppN cap structure by the eukaryotic initiation factor 4F (eIF4F), a heterotrimeric complex consisting of the cap-binding protein, eIF4E, along with eIF4G, and eIF4A. The eIF4E-binding proteins (4E-BPs) repress translation by disrupting eIF4F formation, thereby preventing ribosome recruitment to the mRNA. Of the three 4E-BPs, 4E-BP2 is the predominant paralog expressed in the mammalian brain and plays an important role in synaptic plasticity and learning and memory. 4E-BP2 undergoes asparagine deamidation, solely in the brain, during early postnatal development. Deamidation spontaneously converts asparagines into a mixture of aspartates or isoaspartates, the latter of which may be destabilizing to proteins. The enzyme protein l-isoaspartyl methyltransferase (PIMT) prevents isoaspartate accumulation by catalyzing the conversion of isoaspartates to aspartates. PIMT exhibits high activity in the brain, relative to other tissues. We report here that 4E-BP2 is a substrate for PIMT. In vitro deamidated 4E-BP2 accrues isoapartyl residues and is methylated by recombinant PIMT. Using an antibody that recognizes 4E-BP2, which harbors isoaspartates at the deamidation sites, Asn99 and Asn102, we demonstrate that 4E-BP2 in PIMT−/− brain lysates contains isoaspartate residues. Further, we show that 4E-BP2 containing isoaspartates lacks the augmented association with raptor that is a feature of deamidated 4E-BP2.

Mextli Is a Novel Eukaryotic Translation Initiation Factor 4E-Binding Protein That Promotes Translation in Drosophila melanogaster

ABSTRACT Translation is a fundamental step in gene expression, and translational control is exerted in many developmental processes. Most eukaryotic mRNAs are translated by a cap-dependent mechanism, which requires recognition of the 5′-cap structure of the mRNA by eukaryotic translation initiation factor 4E (eIF4E). eIF4E activity is controlled by eIF4E-binding proteins (4E-BPs), which by competing with eIF4G for eIF4E binding act as translational repressors. Here, we report the discovery of Mextli (Mxt), a novel Drosophila melanogaster 4E-BP that in sharp contrast to other 4E-BPs, has a modular structure, binds RNA, eIF3, and several eIF4Es, and promotes translation. Mxt is expressed at high levels in ovarian germ line stem cells (GSCs) and early-stage cystocytes, as is eIF4E-1, and we demonstrate the two proteins interact in these cells. Phenotypic analysis of mxt mutants indicates a role for Mxt in germ line stem cell (GSC) maintenance and in early embryogenesis. Our results support the idea that Mxt, like eIF4G, coordinates the assembly of translation initiation complexes, rendering Mxt the first example of evolutionary convergence of eIF4G function.