Multivalent Recruitment of Human Argonaute by GW182.

In miRNA-mediated gene silencing, the physical interaction between human Argonaute (hAgo) and GW182 (hGW182) is essential for facilitating the downstream silencing of the targeted mRNA. GW182 can interact with hAgo via three of the GW/WG repeats in its Argonaute-binding domain: motif-1, motif-2, and the hook motif. The structure of hAgo1 in complex with the hook motif of hGW182 reveals a "gate"-like interaction that is critical for GW182 docking into one of hAgo1's tryptophan-binding pockets. We show that hAgo1 and hAgo2 have a single GW182-binding site and that miRNA binding increases hAgo's affinity to GW182. With target binding occurring rapidly, this ensures that only mature RISC would be recruited for silencing. Finally, we show that hGW182 can recruit up to three copies of hAgo via its three GW motifs. This may explain the observed cooperativity in miRNA-mediated gene silencing.

Multi-domain utilization by TUT4 and TUT7 in control of let-7 biogenesis.

The uridyl transferases TUT4 and TUT7 (collectively called TUT4(7)) switch between two modes of activity, either promoting expression of let-7 microRNA (monoU) or marking it for degradation (oligoU). Lin28 modulates the switch via recruitment of TUT4(7) to the precursor pre-let-7 in stem cells and human cancers. We found that TUT4(7) utilize two multidomain functional modules during the switch from monoU to oligoU. The catalytic module (CM) is essential for both activities, while the Lin28-interacting module (LIM) is indispensable for oligoU. A TUT7 CM structure trapped in the monoU activity staterevealed a duplex-RNA-binding pocket that orients group II pre-let-7 hairpins to favor monoU addition. Conversely, the switch to oligoU requires the ZK domain of Lin28 to drive the formation of a stable ternary complex between pre-let-7 and the inactive LIM. Finally, ZK2 of TUT4(7) aids oligoU addition by engaging the growing oligoU tail through uracil-specific interactions.

PTEN functions by recruitment to cytoplasmic vesicles.

PTEN is proposed to function at the plasma membrane, where receptor tyrosine kinases are activated. However, the majority of PTEN is located throughout the cytoplasm. Here, we show that cytoplasmic PTEN is distributed along microtubules, tethered to vesicles via phosphatidylinositol 3-phosphate (PI(3)P), the signature lipid of endosomes. We demonstrate that the non-catalytic C2 domain of PTEN specifically binds PI(3)P through the CBR3 loop. Mutations render this loop incapable of PI(3)P binding and abrogate PTEN-mediated inhibition of PI 3-kinase/AKT signaling. This loss of function is rescued by fusion of the loop mutant PTEN to FYVE, the canonical PI(3)P binding domain, demonstrating the functional importance of targeting PTEN to endosomal membranes. Beyond revealing an upstream activation mechanism of PTEN, our data introduce the concept of PI 3-kinase signal activation on the vast plasma membrane that is contrasted by PTEN-mediated signal termination on the small, discrete surfaces of internalized vesicles.

Mechanism of Dis3l2 substrate recognition in the Lin28-let-7 pathway.

The pluripotency factor Lin28 inhibits the biogenesis of the let-7 family of mammalian microRNAs. Lin28 is highly expressed in embryonic stem cells and has a fundamental role in regulation of development, glucose metabolism and tissue regeneration. Overexpression of Lin28 is correlated with the onset of numerous cancers, whereas let-7, a tumour suppressor, silences several human oncogenes. Lin28 binds to precursor let-7 (pre-let-7) hairpins, triggering the 3' oligo-uridylation activity of TUT4 and TUT7 (refs 10-12). The oligoU tail added to pre-let-7 serves as a decay signal, as it is rapidly degraded by Dis3l2 (refs 13, 14), a homologue of the catalytic subunit of the RNA exosome. The molecular basis of Lin28-mediated recruitment of TUT4 and TUT7 to pre-let-7 and its subsequent degradation by Dis3l2 is largely unknown. To examine the mechanism of Dis3l2 substrate recognition we determined the structure of mouse Dis3l2 in complex with an oligoU RNA to mimic the uridylated tail of pre-let-7. Three RNA-binding domains form an open funnel on one face of the catalytic domain that allows RNA to navigate a path to the active site different from that of its exosome counterpart. The resulting path reveals an extensive network of uracil-specific interactions spanning the first 12 nucleotides of an oligoU-tailed RNA. We identify three U-specificity zones that explain how Dis3l2 recognizes, binds and processes uridylated pre-let-7 in the final step of the Lin28-let-7 pathway.

The making of a slicer: activation of human Argonaute-1.

Argonautes are the central protein component in small RNA silencing pathways. Of the four human Argonautes (hAgo1-hAgo4) only hAgo2 is an active slicer. We determined the structure of hAgo1 bound to endogenous copurified RNAs to 1.75 Å resolution and hAgo1 loaded with let-7 microRNA to 2.1 Å. Both structures are strikingly similar to the structures of hAgo2. A conserved catalytic tetrad within the PIWI domain of hAgo2 is required for its slicing activity. Completion of the tetrad, combined with a mutation on a loop adjacent to the active site of hAgo1, results in slicer activity that is substantially enhanced by swapping in the N domain of hAgo2. hAgo3, with an intact tetrad, becomes an active slicer by swapping the N domain of hAgo2 without additional mutations. Intriguingly, the elements that make Argonaute an active slicer involve a sophisticated interplay between the active site and more distant regions of the enzyme.

Structural characterization of inhibitors with selectivity against members of a homologous enzyme family.

The aspartate biosynthetic pathway provides essential metabolites for many important biological functions, including the production of four essential amino acids. As this critical pathway is only present in plants and microbes, any disruptions will be fatal to these organisms. An early pathway enzyme, l-aspartate-ß-semialdehyde dehydrogenase, produces a key intermediate at the first branch point of this pathway. Developing potent and selective inhibitors against several orthologs in the l-aspartate-ß-semialdehyde dehydrogenase family can serve as lead compounds for antibiotic development. Kinetic studies of two small molecule fragment libraries have identified inhibitors that show good selectivity against l-aspartate-ß-semialdehyde dehydrogenases from two different bacterial species, Streptococcus pneumoniae and Vibrio cholerae, despite the presence of an identical constellation of active site amino acids in this homologous enzyme family. Structural characterization of enzyme-inhibitor complexes have elucidated different modes of binding between these structurally related enzymes. This information provides the basis for a structure-guided approach to the development of more potent and more selective inhibitors.

The catalytic machinery of a key enzyme in amino Acid biosynthesis.

The aspartate pathway of amino acid biosynthesis is essential for all microbial life but is absent in mammals. Characterizing the enzyme-catalyzed reactions in this pathway can identify new protein targets for the development of antibiotics with unique modes of action. The enzyme aspartate ß-semialdehyde dehydrogenase (ASADH) catalyzes an early branch point reaction in the aspartate pathway. Kinetic, mutagenic, and structural studies of ASADH from various microbial species have been used to elucidate mechanistic details and to identify essential amino acids involved in substrate binding, catalysis, and enzyme regulation. Important structural and functional differences have been found between ASADHs isolated from these bacterial and fungal organisms, opening the possibility for developing species-specific antimicrobial agents that target this family of enzymes.

The structure of a redundant enzyme: a second isoform of aspartate beta-semialdehyde dehydrogenase in Vibrio cholerae.

Aspartate-beta-semialdehyde dehydrogenase (ASADH) is an essential enzyme that is found in bacteria, fungi and plants but not in humans. ASADH produces the first branch-point metabolite in the biosynthetic pathways that lead to the production of lysine, threonine, methionine and isoleucine as well as the cell-wall precursor diaminopimelate. As a consequence, ASADH appears to be an excellent target for the development of novel antibiotics, especially for Gram-negative bacteria that require diaminopimelate for cell-wall biosynthesis. In contrast to the Gram-negative ASADHs, which readily formed well diffracting crystals, the second isoform of aspartate-beta-semialdehyde dehydrogenase from Vibrio cholerae (vcASADH2) was less well behaved in initial crystallization trials. In order to obtain good-quality single crystals of vcASADH2, a buffer-optimization protocol was used in which the initial purification buffer was exchanged into a new condition derived from a pre-crystalline hit. The unliganded structure of vcASADH2 has been determined to 2.2 A resolution to provide additional insight into the structural and functional evolution of the ASADH enzyme family. The overall fold and domain organization of this new structure is similar to the Gram-negative, Gram-positive and archeal ASADH structures determined previously, despite having less than 50% sequence identity to any of these family members. The substrate-complex structure reveals that the binding of L-aspartate-beta-semialdehyde (ASA) to vcASADH2 is accommodated by structural changes in the amino-acid binding site and in the helical subdomain that is involved in the dimer interface. Structural alignments show that this second isoform from Gram-negative V. cholerae most closely resembles the ASADH from a Gram-positive organism and is likely to bind the coenzyme in a different conformation to that observed in the other V. cholerae isoform.

Argonautes confront new small RNAs.

Argonaute is at the heart of all effector complexes in RNA interference. In the classical RNAi pathway Argonaute functions as the Slicer enzyme that cleaves an mRNA target directed by a complementary siRNA. Two recently described Argonaute protein subfamilies mediate distinct functions in RNAi. The Piwi subfamily functions in the germline through a novel class of small RNAs that are longer than Argonaute-specific siRNAs and miRNAs. Piwi-interacting RNAs (piRNAs) carry a 2'-O-methylation on their 3' end and appear to be synthesized by a Piwi Slicer dependent mechanism. Piwi/piRNA complexes in mammals and flies are directly linked to the control of transposable elements during germline development. Amplified RNAi in C. elegans is mediated by secondary siRNAs selectively bound to secondary Argonautes (SAGOs) that belong to a worm-specific Argonaute subfamily (WAGO). Secondary siRNAs are 5' triphosphorylated that may allow specific loading into SAGO complexes that are rate limiting for RNAi in C. elegans. Interestingly, SAGOs lack conserved Slicer amino acid residues and probably act in a Slicer-independent fashion.

The initial step in the archaeal aspartate biosynthetic pathway catalyzed by a monofunctional aspartokinase.

The activation of the beta-carboxyl group of aspartate catalyzed by aspartokinase is the commitment step to amino-acid biosynthesis in the aspartate pathway. The first structure of a microbial aspartokinase, that from Methanococcus jannaschii, has been determined in the presence of the amino-acid substrate L-aspartic acid and the nucleotide product MgADP. The enzyme assembles into a dimer of dimers, with the interfaces mediated by both the N- and C-terminal domains. The active-site functional groups responsible for substrate binding and specificity have been identified and roles have been proposed for putative catalytic functional groups.

Examination of key intermediates in the catalytic cycle of aspartate-beta-semialdehyde dehydrogenase from a gram-positive infectious bacteria.

Aspartate-beta-semialdehyde dehydrogenase (ASADH) catalyzes a critical branch point transformation in amino acid bio-synthesis. The products of the aspartate pathway are essential in microorganisms, and this entire pathway is absent in mammals, making this enzyme an attractive target for antibiotic development. The first structure of an ASADH from a Gram-positive bacterium, Streptococcus pneumoniae, has now been determined. The overall structure of the apoenzyme has a similar fold to those of the Gram-negative and archaeal ASADHs but contains some interesting structural variations that can be exploited for inhibitor design. Binding of the coenzyme NADP, as well as a truncated nucleotide analogue, into an alternative conformation from that observed in Gram-negative ASADHs causes an enzyme domain closure that precedes catalysis. The covalent acyl-enzyme intermediate was trapped by soaking the substrate into crystals of the coenzyme complex, and the structure of this elusive intermediate provides detailed insights into the catalytic mechanism.

A rapid method for the purification of methanol dehydrogenase from Methylobacterium extorquens.

Methanol dehydrogenase (MDH) is a water soluble quinoprotein that catalyzes the oxidation of methanol as an important carbon source in methylotrophic bacteria. A rapid method for the purification of MDH from Methylobacterium extorquens AM1 was developed using a single cation exchange chromatographic step, followed by ultrafiltration for final purification, enzyme concentration, and buffer exchange. MDH was obtained in an excellent overall yield with a final enzyme purity of greater than 97%. Storage at -80 degrees C in 20mM phosphate buffer, pH 7.0, showed only a negligible loss of enzyme activity after six months.

A new branch in the family: structure of aspartate-beta-semialdehyde dehydrogenase from Methanococcus jannaschii.

The structure of aspartate-beta-semialdehyde dehydrogenase (ASADH) from Methanococcus jannaschii has been determined to 2.3 angstroms resolution using multiwavelength anomalous diffraction (MAD) phasing of a selenomethionine-substituted derivative to define a new branch in the family of ASADHs. This new structure has a similar overall fold and domain organization despite less than 10% conserved sequence identity with the bacterial enzymes. However, the entire repertoire of functionally important active site amino acid residues is conserved, suggesting an identical catalytic mechanism but with lower catalytic efficiency. A new coenzyme-binding conformation and dual NAD/NADP coenzyme specificity further distinguish this archaeal branch from the bacterial ASADHs. Several structural differences are proposed to account for the dramatically enhanced thermostability of this archaeal enzyme. Finally, the intersubunit communication channel connecting the active sites in the bacterial enzyme dimer has been disrupted in the archaeal ASADHs by amino acid changes that likely prevent the alternating sites reactivity previously proposed for the bacterial ASADHs.

Structural basis for discrimination between oxyanion substrates or inhibitors in aspartate-beta-semialdehyde dehydrogenase.

The reversible dephosphorylation of beta-aspartyl phosphate to L-aspartate-beta-semialdehyde (ASA) in the aspartate biosynthetic pathway is catalyzed by aspartate-beta-semialdehyde dehydrogenase (ASADH). The phosphate that is present to activate the aspartate carboxyl group is held in a separate and distinct binding site once removed and prior to its release from the enzyme. This site had been shown to be selective for tetrahedral oxyanions, with several competitive inhibitors and alternative substrates previously identified for the reverse reaction. Structural studies have now shown that the most potent oxyanion inhibitor (periodate) and a good alternative substrate (arsenate) each occupy the same catalytic phosphate-binding site. However, a rotation of a threonine side chain (Thr137) in the periodate complex disrupts an important hydrogen-bonding interaction with an active-site glutamate (Glu243) that participates in substrate orientation. This subtle change appears to be the difference between a substrate and an inhibitor of this enzyme.

Critical catalytic functional groups in the mechanism of aspartate-beta-semialdehyde dehydrogenase.

Aspartate-beta-semialdehyde dehydrogenase (ASADH) catalyzes the reductive dephosphorylation of beta-aspartyl phosphate to L-aspartate-beta-semialdehyde in the aspartate biosynthetic pathway. This pathway is not found in humans or other eukaryotic organisms, yet is required for the production of threonine, isoleucine, methionine and lysine in most microorganisms. The mechanism of this enzyme has been examined through the structures of two active-site mutants of ASADH from Haemophilus influenzae. Replacement of the enzyme active-site cysteine with serine (C136S) leads to a dramatic loss of catalytic activity caused by the expected decrease in nucleophilicity, but also by a change in the orientation of the serine hydroxyl group relative to the cysteine thiolate. In contrast, in the H277N active-site mutant the introduced amide is oriented in virtually the same position as that of the histidine imidazole ring. However, a shift in the position of the bound reaction intermediate to accommodate this shorter asparagine side chain, coupled with the inability of this introduced amide to serve as a proton acceptor, results in a 100-fold decrease in the catalytic efficiency of H277N relative to the native enzyme. These mutant enzymes have the same overall fold and high structural identity to native ASADH. However, small perturbations in the positioning of essential catalytic groups or reactive intermediates have dramatic effects on catalytic efficiency.

The role of substrate-binding groups in the mechanism of aspartate-beta-semialdehyde dehydrogenase.

The reversible dephosphorylation of beta-aspartyl phosphate to L-aspartate-beta-semialdehyde (ASA) in the aspartate biosynthetic pathway is catalyzed by aspartate-beta-semialdehyde dehydrogenase (ASADH). The product of this reaction is a key intermediate in the biosynthesis of diaminopimelic acid, an integral component of bacterial cell walls and a metabolic precursor of lysine and also a precursor in the biosynthesis of threonine, isoleucine and methionine. The structures of selected Haemophilus influenzae ASADH mutants were determined in order to evaluate the residues that are proposed to interact with the substrates ASA or phosphate. The substrate Km values are not altered by replacement of either an active-site arginine (Arg270) with a lysine or a putative phosphate-binding group (Lys246) with an arginine. However, the interaction of phosphate with the enzyme is adversely affected by replacement of Arg103 with lysine and is significantly altered when a neutral leucine is substituted at this position. A conservative Glu243 to aspartate mutant does not alter either ASA or phosphate binding, but instead results in an eightfold increase in the Km for the coenzyme NADP. Each of the mutations is shown to cause specific subtle active-site structural alterations and each of these changes results in decreases in catalytic efficiency ranging from significant (approximately 3% native activity) to substantial (<0.1% native activity).

Purification and preliminary characterization of brain aspartoacylase.

Aspartoacylase catalyzes the deacetylation of N-acetylaspartic acid (NAA) in the brain to produce acetate and L-aspartate. An aspartoacylase deficiency, with concomitant accumulation of NAA, is responsible for Canavan disease, a lethal autosomal recessive disorder. To examine the mechanism of this enzyme the genes encoding murine and human aspartoacylase were cloned and expressed in Escherichia coli. A significant portion of the enzyme is expressed as soluble protein, with the remainder found as inclusion bodies. A convenient enzyme-coupled continuous spectrophotometric assay has been developed for measuring aspartoacylase activity. Kinetic parameters were determined with the human enzyme for NAA and for selected N-acyl analogs that demonstrate relaxed substrate specificity with regard to the nature of the acyl group. The clinically relevant E285A mutant reveals an altered enzyme with poor stability and barely detectable activity, while a more conservative E285D substitution leads to only fivefold lower activity than native aspartoacylase.