Crystal structure and ligand binding of the MID domain of a eukaryotic Argonaute protein

Argonaute (AGO) proteins are core components of RNA‐induced silencing complexes and have essential roles in RNA‐mediated gene silencing. They are characterized by a bilobal architecture, consisting of one lobe containing the amino‐terminal and PAZ domains and another containing the MID and PIWI domains. Except for the PAZ domain, structural information on eukaryotic AGO domains is not yet available. In this study, we report the crystal structure of the MID domain of the eukaryotic AGO protein QDE‐2 from Neurospora crassa. This domain adopts a Rossmann‐like fold and recognizes the 5′‐terminal nucleotide of a guide RNA in a manner similar to its prokaryotic counterparts. The 5′‐nucleotide‐binding site shares common residues with a second, adjacent ligand‐binding site, suggesting a mechanism for the cooperative binding of ligands to the MID domain of eukaryotic AGOs.     

Argonaute MID domain takes centre stage

Two recent papers, one in EMBO reports and one in Nature give us the first eukaryotic structures of Argonaute MID domains; providing a structural basis for the 5′-nucleotide recognition of the guide strand and a possible explanation for the allosteric regulation of RNA binding.EMBO Rep (2010) advance online publication. doi: 10.1038/embor.2010.81Argonaute (AGO) proteins are the central component of small RNA-mediated gene silencing in eukaryotes. Functional AGO complexes are loaded with single-stranded small RNAs, which guide AGO to a messenger RNA (mRNA) target through base pairing. Although the structure of a full-length eukaryotic AGO has yet to be described, insights into the mechanism of guide RNA binding and target recognition have been revealed by the structures of distantly related AGO homologues from archaea and eubacteria (Song et al, 2004; Wang et al, 2008, 2009). These studies show that AGO proteins are composed of amino-terminal, PAZ (PIWI/Argonaute/Zwille), MID (middle) and PIWI (P-element-induced whimpy testes) domains. The phosphorylated 5′-end of the guide strand RNA is localized in the MID–PIWI domain interface with the 3′-end anchored to the PAZ domain. On binding to mRNA the catalytic RNase H-like active site located in the PIWI domain is in position to cleave the targeted mRNA.Two recent papers, one in EMBO reports (Boland et al, 2010) and one in Nature (Frank et al, 2010), give us the first eukaryotic structures of AGO MID domains. The human AGO MID domain structure provides a structural basis for the 5′-nucleotide recognition of the guide strand observed in eukaryotic AGOs, and the structure of the MID domain of QDE-2 from Neurospora crassa published in this journal offers a possible explanation for the allosteric regulation of RNA binding discovered earlier this year by Rachel Green''s group (Djuranovic et al, 2010)The two structures have a similar topology resembling a Rossmann-like fold with four β-strands forming a central β-sheet flanked by α-helices. Superposition of the two structures, which are 24% identical in sequence, shows that they are also similar in three dimensions, with a root mean square deviation (r.m.s.d.) of 2.1 Å. The archaeal and eubacterial AGO MID domains solved previously share less than 20% sequence identity and have a greater than 2.5 Å r.m.s.d. for backbone atoms from both QDE-2 and human AGO2, despite having a similar overall fold.Crystals of the QDE-2 MID domain contain two sulphate ions. The first sulphate (sulphate I) is coordinated by the highly conserved amino acids Tyr 595, Lys 599 and Lys 638, and is in the same position as the 5′-phosphate of UMP observed in the human AGO2 MID domain structure (Fig 1A). These interactions are similar to those observed for the 5′-phosphate of the guide strand of the previously solved archaeal and eubacterial structures (Ma et al, 2005; Parker et al, 2005; Wang et al, 2008). Thus, sulphate I bound in the QDE-2 MID domain structure likely represents the 5′-nucleotide-binding site. Most intriguing is the position of the second sulphate (sulphate II), located in an adjacent but partly overlapping binding site with sulphate I. Sulphate II is 6.3 Å from sulphate I, shares coordination with Lys 599 and Lys 638, and is further coordinated by Thr 610. Sulphate II can be excluded from representing the phosphate backbone of a microRNA (miRNA) or target because it is bound in the side of the MID domain opposite from where the guide RNA extends from the 5′-nucleotide-binding site. Although the presence of sulphate II does not guarantee a biologically relevant ligand-binding site, it is tempting to speculate, in the light of a recent study by Djuranovic et al, that sulphate II occupies an allosteric ligand-binding site.Open in a separate windowFigure 1Structural comparison of Neurospora crassa QDE-2 MID domain and human Argonaute 2 MID domain. (A) The N. crassa QDE-2 MID domain structure (green ribbon). UMP is modelled from a superposition of the human AGO2 MID domain structure in a complex with UMP (Protein Data Bank code 3LUJ). Sulphate I and II (S I and S II) are shown as observed in the QDE-2 structure. Conserved QDE-2 amino acids involved in binding sulphate I and II are shown as sticks. (B) Human AGO2 MID domain structure (blue ribbon) in complex with UMP (sticks). The nucleotide specificity loop is coloured in yellow. Sulphate I and II are modelled from a superposition of the N. crassa QDE-2 MID domain. AGO, Argonaute; MID, middle.Djuranovic et al describe a second ligand-binding site in the Drosophila melanogaster AGO1 MID domain that is separate and distinct from the 5′-nucleotide-binding site. They demonstrate that free nucleotides, including the cap analogue m⁷GpppG, bind to an allosteric site, which in turn enhances the binding of miRNA. Cap binding was reported previously for human AGO2 (Kiriakidou et al, 2007), leading to the proposal that two phenylalanine residues in the MID domain make stacking interactions with the m⁷GpppG cap structure, analogous to eukaryotic initiation factor 4E. However, in the human AGO2 MID domain structure it is clear that these phenylalanine residues are on opposite sides of the MID domain and are located in the hydrophobic core. In the QDE-2 MID domain only one of these phenylalanines is conserved, but a similar conclusion is drawn on the basis of the positions of the two residues being more than 25 Å apart. This strongly argues that an alternative mechanism exists for cap binding by eukaryotic AGO proteins.The data presented by Djuranovic et al might be explained by the structure of the QDE-2 MID domain, with sulphate I representing the 5′-binding site of a miRNA and sulphate II representing the allosteric site. This argument is strengthened by the fact that the two binding sites are partly shared, namely by interactions with the side chains of Lys 599 and Lys 638, so it would not be surprising that binding of a ligand to one site would have a positive effect on ligand binding at the other site. To identify the location of the potential allosteric site, Djuranovic et al mutated Asp 627—a conserved residue located in a loop 15 Å away from the 5′-nucleotide binding site—to a lysine in D. melanogaster AGO1. The D627K mutant failed to bind cap analogues, indicating the importance of Asp 627 for binding ligands in the allosteric site. When mapped onto the new structures of the QDE-2 and human AGO2 MID domains, this loop and Asp 627 (Asp 603 in QDE-2 and Asp 537 in human AGO2) are in the vicinity of sulphate II, thus Asp 627 is probably a part of the allosteric binding site (Fig 1A,B). The most significant finding in the Djuranovic et al study is that the D627K mutant located in the allosteric site fails to bind to miRNA in the 5′-nucleotide-binding site and no longer associates with GW182, an essential factor in miRNA-induced gene silencing.Almost all miRNA sequences and RNA sequencing data obtained from immunopurified AGO proteins show a marked bias for uridine and adenosine nucleotides at the 5′-end of miRNA guide strands. The structure of human AGO2 MID domain alone and in a complex with UMP, AMP, GMP and CMP provides the first explanation for the observed 5′-nucleotide bias in eukaryotic AGO proteins (Frank et al, 2010). There is little movement induced on nucleotide binding in the overall fold of the MID domain. Electron density is observed for the entire nucleotide in the case of UMP and AMP. The 5′-phosphate in the UMP and AMP complexes is hydrogen bonded to the highly conserved side chains of Tyr 529, Lys 533, Gln 545 and Lys 570. The base of each nucleotide stacks with Tyr 529, completing a nonspecific recognition pocket for the 5′-nucleotide (Fig 1B). A similar pocket is formed in the N. crassa MID domain structure to recognize the 5′-nucleotide. Interestingly, clear electron density for only the phosphate and ribose is observed for GMP and CMP, with density for the GMP and CMP bases missing. These results are consistent with the preference for UMP and AMP binding to human AGO2, but where does this specificity originate?A closer look at the UMP and AMP complex structures show that base-specific contacts are formed with backbone atoms of a loop spanning residues Pro 523 through Pro 527, appropriately termed the nucleotide specificity loop. In the case of GMP and CMP, the hydrogen-bonding partners are in the opposite orientation, resulting in charge repulsion from backbone atoms in the nucleotide specificity loop, thus explaining the observed bias in the 5′-position of the guide strand. In the nucleotide-free structure, the conformation of the nucleotide specificity loop is merely unchanged, suggesting that the loop is rather rigid. When the length of the nucleotide specificity loop is increased by the insertion of a single glycine residue, the specificity for uridine and adenosine nucleotides is lost, further endorsing the idea that the particular conformation and rigid nature of the loop is essential for specific base recognition. Interestingly, the QDE-2 MID domain deviates from the human AGO2 MID domain in the nucleotide specificity loop. An insertion of an aspartate residue in QDE-2 makes the nucleotide specificity loop one amino acid longer, suggesting that QDE-2 might have lost its specificity for nucleotides at the 5′-end of miRNAs, although this is yet to be tested.A complete understanding of miRNA loading and the allosteric mechanism will have to await structures of full-length eukaryotic AGO proteins, as the PIWI domain contributes numerous contacts with the MID domain, encompassing both the 5′-nucleotide-binding site and the putative allosteric site. However, the structures of N. crassa QDE-2 MID and human AGO2 MID domains together are important pieces of the puzzle in our understanding of the mechanism of RNA interference. Specific recognition of the 5′-nucleotide of the guide strand might be a quality control mechanism for some eukaryotic AGOs, ensuring that after primary processing the correct miRNA guide sequence is loaded. Once loaded with a proper guide strand, AGO might trigger the adjacent allosteric site to bind to m⁷GpppG-capped mRNA, GW182 or other unknown ligands. Together, these events ultimately lead to effective gene silencing.     

Mechanical transduction mechanisms of RecA-like molecular motors

A majority of ATP-dependent molecular motors are RecA-like proteins, performing diverse functions in biology. These RecA-like molecular motors consist of a highly conserved core containing the ATP-binding site. Here I examined how ATP binding within this core is coupled to the conformational changes of different RecA-like molecular motors. Conserved hydrogen bond networks and conformational changes revealed two major mechanical transduction mechanisms: (1) intra-domain conformational changes and (2) inter-domain conformational changes. The intra-domain mechanism has a significant hydrogen bond rearrangement within the domain containing the P-loop, causing relative motion between two parts of the protein. The inter-domain mechanism exhibits little conformational change in the P-loop domain. Instead, the major conformational change is observed between the P-loop domain and an adjacent domain or subunit containing the arginine finger. These differences in the mechanical transduction mechanisms may link to the underlying energy surface governing a Brownian ratchet or a power stroke.     

Evolutionary conservation of a unique amino acid sequence in human DICER protein essential for binding to Argonaute family proteins

The Argonaute family and DICER proteins are major key proteins involved in the RNA-mediated gene silencing mechanism of various species. In this mechanism, cleavage of messenger RNAs (mRNA) or suppression of mRNA translation takes place via small RNAs that are uniquely processed by DICER. Previously, we demonstrated that human Argonaute family proteins bind to DICER. In this study, we identified a unique amino acid sequence of 127 amino acids in the RIBOc-A domain of human DICER protein as a "binding site" to Argonaute proteins. Comparative genomics analysis revealed that this unique amino acid sequence is highly conserved in the vertebrates, but not found in the non-vertebrate species. Significant difference in the RIBOc-A domain of DICER protein between vertebrate and non-vertebrate species may help exploring the functional complexity in the RNA-mediated gene silencing mechanism.     

Genome wide cloning of maize meiotic recombinase Dmc1 and its functional structure through molecular phylogeny

The development of meiotic division and associated genetic recombination paved the way for evolutionary changes. However, the secondary and tertiary structure and functional domains of many of the proteins involved in genetic recombination have not been studied in detail. We used the human Dmc1 gene product along with secondary and tertiary domain structures of Escherichia coli RecA protein to help determine the molecular structure and function of maize Dmc1, which is required for synaptonemal complex formation and cell cycle progression. The maize recombinase Dmc1 gene was cloned and characterized, using rice Dmc1 cDNA as an orthologue. The deduced amino acid sequence was used for elaborating its 3-D structure, and functional analysis was made with the CDD software, showing significant identity of the Dmc1 gene product in Zea mays with that of Homo sapiens. Based on these results, the domains and motives of WalkerA and WalkerB as ATP binding sites, a multimer site (BRC) interface, the putative ssDNA binding L1 and L2 loops, the putative dsDNA binding helix-hairpin-helix, a polymerization motif, the subunit rotation motif, and a small N-terminal domain were proposed for maize recombinase Dmc1.     

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.     

Evolution of plant Ash1 SET genes: structural divergence and functional differentiation

Plant Ash1 SET proteins are involved in H3K36 methylation, and play a key role in plant reproductive development. Genes encoding Ash1 SET proteins constitute a multigene family in which the copy number varies among plant species and functional divergence appears to have occurred repeatedly. To investigate the evolutionary history and functional differentiation of the Ash1 SET gene family, we made a comprehensive evolutionary analysis of this gene family from eleven major representatives of green plants. A novel deep sister relationship grouping previously resolved II-1 and II-2 orthologous groups was identified. The absence of AWS domain in the group II-2 suggests that the independent losses of AWS domain have occurred during evolution. A diversity of gene structures in plant Ash1 SET gene family have been presented since the divergence of Physcomitrella patens (moss) from the other land plants. A small proportion of codons in SET domain regions were detected to be under positive selection along the branches ancestral to land plant and angiosperms, which may have allowed changes of substrate specificity among different evolutionary groups while maintaining the primary function of SET domains. Our predictive subcellular localization and comparative anatomical meta-expression analyses can assort with the structural divergences of Ash1 SET proteins.     

The evolution of extracellular fibrillins and their functional domains

Fibrillins constitute the major backbone of multifunctional microfibrils in elastic and non-elastic extracellular matrices, and are known to interact with several binding partners including tropoelastin and integrins. Here, we study the evolution of fibrillin proteins. Following sequence collection from 39 organisms representative of the major evolutionary groups, molecular evolutionary genetics and phylogeny inference software were used to generate a series of evolutionary trees using distance-based and maximum likelihood methods. The resulting trees support the concept of gene duplication as a means of generating the three vertebrate fibrillins. Beginning with a single fibrillin sequence found in invertebrates and jawless fish, a gene duplication event, which coincides with the appearance of elastin, led to the creation of two genes. One of the genes significantly evolved to become the gene for present-day fibrillin-1, while the other underwent evolutionary changes, including a second duplication, to produce present-day fibrillin-2 and fibrillin-3. Detailed analysis of several sequences and domains within the fibrillins reveals distinct similarities and differences across various species. The RGD integrin-binding site in TB4 of all fibrillins is conserved in cephalochordates and vertebrates, while the integrin-binding site within cbEGF18 of fibrillin-3 is a recent evolutionary change. The proline-rich domain in fibrillin-1, glycine-rich domain in fibrillin-2 and proline-/glycine-rich domain in fibrillin-3 are found in all analyzed tetrapod species, whereas it is completely replaced with an EGF-like domain in cnidarians, arthropods, molluscs and urochordates. All collected sequences contain the first 9-cysteine hybrid domain, and the second 8-cysteine hybrid domain with exception of arthropods containing an atypical 10-cysteine hybrid domain 2. Furin cleavage sites within the N- and C-terminal unique domains were found for all analyzed fibrillin sequences, indicating an essential role for processing of the fibrillin pro-proteins. The four cysteines in the unique N-terminus and the two cysteines in the unique C-terminus are also highly conserved.     

Sequence-specific DNA recognition by the myb-like domain of the human telomere binding protein TRF1: a model for the protein-DNA complex.

Telomeres consist of tandem arrays of short G-rich sequence motifs packaged by specific DNA binding proteins. In humans the double-stranded telomeric TTAGGG repeats are specifically bound by TRF1 and TRF2. Although telomere binding proteins from evolutionarily distant species are not sequence homologues, they share a Myb-like DNA binding motif. Here we have used gel retardation, primer extension and DNase I footprinting analyses to define the binding site of the isolated Myb-like domain of TRF1 and present a three-dimensional model for its interaction with human telomeric DNA. Our results suggest that the Myb-like domain of TRF1 recognizes a binding site centred on the sequence GGGTTA and that its DNA binding mode is similar to that of the homeodomain-like motifs of the yeast telomere binding protein RAP1. The implications of these findings for recognition of telomeric DNA in general are discussed.     

All in the family: structural and evolutionary relationships among three modular proteins with diverse functions and variable assembly

The crystal structures of three proteins of diverse function and low sequence similarity were analyzed to evaluate structural and evolutionary relationships. The proteins include a bacterial bleomycin resistance protein, a bacterial extradiol dioxygenase, and human glyoxalase I. Structural comparisons, as well as phylogenetic analyses, strongly indicate that the modern family of proteins represented by these structures arose through a rich evolutionary history that includes multiple gene duplication and fusion events. These events appear to be historically shared in some cases, but parallel and historically independent in others. A significant early event is proposed to be the establishment of metal-binding in an oligomeric ancestor prior to the first gene fusion. Variations in the spatial arrangements of homologous modules are observed that are consistent with the structural principles of three-dimensional domain swapping, but in the unusual context of the formation of larger monomers from smaller dimers or tetramers. The comparisons support a general mechanism for metalloprotein evolution that exploits the symmetry of a homooligomeric protein to originate a metal binding site and relies upon the relaxation of symmetry, as enabled by gene duplication, to establish and refine specific functions.     

Arabidopsis Argonaute 2 regulates innate immunity via miRNA393(∗)-mediated silencing of a Golgi-localized SNARE gene, MEMB12

Argonaute (AGO) proteins are critical components of RNA silencing pathways that bind small RNAs and mediate gene silencing at their target sites. We found that Arabidopsis AGO2 is highly induced by the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). Further genetic analysis demonstrated that AGO2 functions in antibacterial immunity. One abundant species of AGO2-bound small RNA is miR393b(?), which targets a Golgi-localized SNARE gene, MEMB12. Pst infection downregulates MEMB12 in a miR393b(?)-dependent manner. Loss of function of MEMB12, but not SYP61, another intracellular SNARE, leads to increased exocytosis of an antimicrobial pathogenesis-related protein, PR1. Overexpression of miR393b(?) resembles memb12 mutant in resistance responses. Thus, AGO2 functions in antibacterial immunity by binding miR393b(?) to modulate exocytosis of antimicrobial PR proteins via MEMB12. Since miR393 also contributes to antibacterial responses, miR393(?)/miR393 represent an example of a miRNA(?)/miRNA pair that functions in immunity through two distinct AGOs: miR393(?) through AGO2 and miR393 through AGO1.     

Lessons on RNA Silencing Mechanisms in Plants from Eukaryotic Argonaute Structures

RNA silencing refers to a collection of gene regulatory mechanisms that use small RNAs for sequence specific repression. These mechanisms rely on ARGONAUTE (AGO) proteins that directly bind small RNAs and thereby constitute the central component of the RNA-induced silencing complex (RISC). AGO protein function has been probed extensively by mutational analyses, particularly in plants where large allelic series of several AGO proteins have been isolated. Structures of entire human and yeast AGO proteins have only very recently been obtained, and they allow more precise analyses of functional consequences of mutations obtained by forward genetics. To a large extent, these analyses support current models of regions of particular functional importance of AGO proteins. Interestingly, they also identify previously unrecognized parts of AGO proteins with profound structural and functional importance and provide the first hints at structural elements that have important functions specific to individual AGO family members. A particularly important outcome of the analysis concerns the evidence for existence of Gly-Trp (GW) repeat interactors of AGO proteins acting in the plant microRNA pathway. The parallel analysis of AGO structures and plant AGO mutations also suggests that such interactions with GW proteins may be a determinant of whether an endonucleolytically competent RISC is formed.     

Divalent cations stabilize the alpha 1 beta 1 integrin I domain.

Recent structural and functional analyses of alpha integrin subunit I domains implicate a region in cation and ligand binding referred to as the metal ion-dependent adhesion site (MIDAS). Although the molecular interactions between Mn2+ and Mg2+ and the MIDAS region have been defined by crystallographic analyses, the role of cation in I domain function is not well understood. Recombinant alpha 1 beta 1 integrin I domain (alpha1-I domain) binds collagen in a cation-dependent manner. We have generated and characterized a panel of antibodies directed against the alpha1-I domain, and selected one (AJH10) that blocks alpha 1 beta 1 integrin function for further study. The epitope of AJH10 was localized within the loop between the alpha 3 and alpha 4 helices which contributes one of the metal coordination sites of the MIDAS structure. Kinetic analyses of antibody binding to the I domain demonstrate that divalent cation is required to stabilize the epitope. Denaturation experiments demonstrate that cation has a dramatic effect on the stabilization of the I domain structure. Mn2+ shifts the point at which the I domain denatures from 3.4 to 6.3 M urea in the presence of the denaturant, and from 49.5 to 58.6 degrees C following thermal denaturation. The structural stability provided to the alpha1-I domain by divalent cations may contribute to augmented ligand binding that occurs in the presence of these cations.