X-ray crystal structures of the oxidized and reduced forms of the rubredoxin from the marine hyperthermophilic archaebacterium Pyrococcus furiosus.

The structures of the oxidized and reduced forms of the rubredoxin from the archaebacterium, Pyrococcus furiosus, an organism that grows optimally at 100 degrees C, have been determined by X-ray crystallography to a resolution of 1.8 A. Crystals of this rubredoxin grow in space group P2(1)2(1)2(1) with room temperature cell dimensions a = 34.6 A, b = 35.5 A, and c = 44.4 A. Initial phases were determined by the method of molecular replacement using the oxidized form of the rubredoxin from the mesophilic eubacterium, Clostridium pasteurianum, as a starting model. The oxidized and reduced models of P. furiosus rubredoxin each contain 414 nonhydrogen protein atoms comprising 53 residues. The model of the oxidized form contains 61 solvent H2O oxygen atoms and has been refined with X-PLOR and TNT to a final R = 0.178 with root mean square (rms) deviations from ideality in bond distances and bond angles of 0.014 A and 2.06 degrees, respectively. The model of the reduced form contains 37 solvent H2O oxygen atoms and has been refined to R = 0.193 with rms deviations from ideality in bond lengths of 0.012 A and in bond angles of 1.95 degrees. The overall structure of P. furiosus rubredoxin is similar to the structures of mesophilic rubredoxins, with the exception of a more extensive hydrogen-bonding network in the beta-sheet region and multiple electrostatic interactions (salt bridge, hydrogen bonds) of the Glu 14 side chain with groups on three other residues (the amino-terminal nitrogen of Ala 1; the indole nitrogen of Trp 3; and the amide nitrogen group of Phe 29). The influence of these and other features upon the thermostability of the P. furiosus protein is discussed.     

Crystal structure of Apaf-1 caspase recruitment domain: an alpha-helical Greek key fold for apoptotic signaling.

The caspase recruitment domain (CARD) of Apaf-1 binds to the CARD of caspase-9 to trigger a proteolytic cascade that leads to apoptotic cell death. We report the crystal structure of the Apaf-1 CARD at 1. 3 A resolution, solved in a two-element multiwavelength anomalous dispersion (MAD) X-ray diffraction experiment. This CARD adopts a six-helix bundle fold with Greek key topology surrounding an extensive hydrophobic core. This fold, which we call the "death fold", is found in other domains that mediate interactions in apoptotic signaling despite very low sequence identity. From a structure-based alignment, we identify conserved patterns that characterize the death fold and its subclasses. Like the Ig-fold, it provides a rigid structural scaffold upon which diverse recognition surfaces are assembled.     

Crystal structures of two intermediates in the assembly of the papillomavirus replication initiation complex

Initiation of DNA replication of the papillomavirus genome is a multi-step process involving the sequential loading of viral E1 protein subunits onto the origin of replication. Here we have captured structural snapshots of two sequential steps in the assembly process. Initially, an E1 dimer binds to adjacent major grooves on one face of the double helix; a second dimer then binds to another face of the helix. Each E1 monomer has two DNA-binding modules: a DNA-binding loop, which binds to one DNA strand and a DNA-binding helix, which binds to the opposite strand. The nature of DNA binding suggests a mechanism for the transition between double- and single-stranded DNA binding that is implicit in the progression to a functional helicase.     

The DNA-binding domain of human papillomavirus type 18 E1. Crystal structure, dimerization, and DNA binding

High risk types of human papillomavirus, such as type 18 (HPV-18), cause cervical carcinoma, one of the most frequent causes of cancer death in women worldwide. DNA replication is one of the central processes in viral maintenance, and the machinery involved is an excellent target for the design of antiviral therapy. The papillomaviral DNA replication initiation protein E1 has origin recognition and ATP-dependent DNA melting and helicase activities, and it consists of a DNA-binding domain and an ATPase/helicase domain. While monomeric in solution, E1 binds DNA as a dimer. Dimerization occurs via an interaction of hydrophobic residues on a single alpha-helix of each monomer. Here we present the crystal structure of the monomeric HPV-18 E1 DNA-binding domain refined to 1.8-A resolution. The structure reveals that the dimerization helix is significantly different from that of bovine papillomavirus type 1 (BPV-1). However, we demonstrate that the analogous residues required for E1 dimerization in BPV-1 and the low risk HPV-11 are also required for HPV-18 E1. We also present evidence that the HPV-18 E1 DNA-binding domain does not share the same nucleotide and amino acid requirements for specific DNA recognition as BPV-1 and HPV-11 E1.     

FUS Regulates Activity of MicroRNA-Mediated Gene Silencing

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Slicer and the argonautes

Though they started out as somewhat mysterious components of the RNAi effector complexes, Argonaute proteins have since taken center stage in RNAi gene silencing. They interact with small RNAs to effect gene silencing in all RNAi-related pathways known so far. We will review the dramatic advances in our understanding of the role of the Argonautes in RNAi through studies of their structure and function.     

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.     

The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes

RISC, the RNA-induced silencing complex, uses short interfering RNAs (siRNAs) or micro RNAs (miRNAs) to select its targets in a sequence-dependent manner. Key RISC components are Argonaute proteins, which contain two characteristic domains, PAZ and PIWI. PAZ is highly conserved and is found only in Argonaute proteins and Dicer. We have solved the crystal structure of the PAZ domain of Drosophila Argonaute2. The PAZ domain contains a variant of the OB fold, a module that often binds single-stranded nucleic acids. PAZ domains show low-affinity nucleic acid binding, probably interacting with the 3' ends of single-stranded regions of RNA. PAZ can bind the characteristic two-base 3' overhangs of siRNAs, indicating that although PAZ may not be a primary nucleic acid binding site in Dicer or RISC, it may contribute to the specific and productive incorporation of siRNAs and miRNAs into the RNAi pathway.