Paul J. McLaughlin

Latrunculin alters the actin-monomer subunit interface to prevent polymerization

atrunculin-A is a drug that is capable of rapidly, reversibly and specifically disrupting the actin cytoskeleton. The efficacy of its action has made it a compound of choice in many cell-biology laboratories, supplanting the classic actin-depolymerizing drug cytochalasin-D. One reason for this is that the mode of action of latrunculin seems to be less complex than that of cytochalasin. Whereas the latter affects the kinetics of actin-filament polymerization at both the barbed and pointed ends, latrunculin-A seems to associate only with actin monomers, thereby preventing them from repolymerizing into filaments. The association of latrunculin with monomeric, rather than filamentous, actin gave us the opportunity to further our understanding of this interaction by detailed structural analysis of actin monomers using crystallographic techniques. Here we show the first high-resolution structure of an actin-disrupting drug in association with actin and discuss how its interactions with actin, and the conformational changes that its binding causes, may explain its mode of action within the cell. Latrunculin (Fig. 1a) is purified from Latrunculia magnificans, a Red Sea sponge that exudes a noxious, red fluid that kills fish within minutes. Two related compounds, latrunculin-A and latrunculinB, isolated from the fluid were shown to depolymerize actin structures both in vitro and in vivo. The in vitro studies showed that latrunculin binds only to the actin monomer and that the kinetics of this interaction are consistent with the complex being unable to polymerize. Unlike cytochalasin, latrunculin can disrupt the actin cytoskeleton in yeast cells. This has enabled genetic studies to be carried out that have facilitated the identification of point mutations in the actin gene that cause cells to become resistant to the effects of the drug (Fig. 1b). The mutations that give rise to latrunculin resistance were found to be clustered around a distinct site, close to the nucleotide-binding site, which indicated that they might identify a potential binding site for latrunculin. However, as this site is not close to recognized subunit contacts in the filament, or to known binding sites for other proteins that associate with actin, the mechanism by which latrunculin exerts its effects has remained unclear. Actin has never been known to crystallize in the absence of a binding protein that keeps it in a monodispersed state. Of the three known examples of such binding proteins, profilin is inappropriate as it promotes nucleotide exchange, whereas deoxyribonuclease1 binds to domains that have been implicated, in studies of yeast genetics, in latrunculin binding. In contrast, gelsolin domain 1 in complex with actin leaves these domains free and also reduces nucleotide exchange, as does latrunculin. We therefore soaked latrunculin-A L

Mutations in ADAR1 cause Aicardi-Goutières syndrome associated with a type I interferon signature

Adenosine deaminases acting on RNA (ADARs) catalyze the hydrolytic deamination of adenosine to inosine in double-stranded RNA (dsRNA) and thereby potentially alter the information content and structure of cellular RNAs. Notably, although the overwhelming majority of such editing events occur in transcripts derived from Alu repeat elements, the biological function of non-coding RNA editing remains uncertain. Here, we show that mutations in ADAR1 (also known as ADAR) cause the autoimmune disorder Aicardi-Goutières syndrome (AGS). As in Adar1-null mice, the human disease state is associated with upregulation of interferon-stimulated genes, indicating a possible role for ADAR1 as a suppressor of type I interferon signaling. Considering recent insights derived from the study of other AGS-related proteins, we speculate that ADAR1 may limit the cytoplasmic accumulation of the dsRNA generated from genomic repetitive elements.

The crystal structure of plasma gelsolin: implications for actin severing, capping, and nucleation.

The structure of gelsolin has been determined by crystallography and comprises six structurally related domains that, in a Ca2+-free environment, pack together to form a compact globular structure in which the putative actin-binding sequences are not sufficiently exposed to enable binding to occur. We propose that binding Ca2+ can release the connections that join the N- and C-terminal halves of gelsolin, enabling each half to bind actin relatively independently. Domain shifts are proposed in response to Ca2+ as bases for models of how gelsolin acts to sever, cap, or nucleate F-actin filaments. The structure also invites discussion of polyphosphoinositide binding to segment 2 and suggests how mutation at Asp-187 could initiate a series of events that lead to deposition of amyloid plaques, as observed in victims of familial amyloidosis (Finnish type).

Dimerisation of a chromo shadow domain and distinctions from the chromodomain as revealed by structural analysis

BACKGROUND Proteins such as HP1, found in fruit flies and mammals, and Swi6, its fission yeast homologue, carry a chromodomain (CD) and a chromo shadow domain (CSD). These proteins are required to form functional transcriptionally silent centromeric chromatin, and their mutation leads to chromosome segregation defects. CSDs have only been found in tandem in proteins containing the related CD. Most HP1-interacting proteins have been found to associate through the CSD and many of these ligands contain a conserved pentapeptide motif. RESULTS The 1.9 A crystal structure of the Swi6 CSD is presented here. This reveals a novel dimeric structure that is distinct from the previously reported monomeric nuclear magnetic resonance (NMR) structure of the CD from the mouse modifier 1 protein (MoMOD1, also known as HP1beta or M31). A prominent pit with a non-polar base is generated at the dimer interface, and is commensurate with binding an extended pentapeptide motif. Sequence alignments based on this structure highlight differences between CDs and CSDs that are superimposed on a common structural core. The analyses also revealed a previously unrecognised circumferential hydrophobic sash around the surface of the CD structure. CONCLUSIONS Dimerisation through the CSD of HP1-like proteins results in the simultaneous formation of a putative protein-protein interaction pit, providing a potential means of targeting CSD-containing proteins to particular chromatin sites.

Methylation: lost in hydroxylation?

Methylation of histone tails is a key determinant in forming active and silent states of chromatin. Histone methylation was regarded as irreversible until the recent identification of a lysine‐specific histone demethylase (LSD1), which acts specifically on mono‐ and dimethylated histone H3 lysine 4. Here, we propose that the fission yeast protein Epe1 is a putative histone demethylase that could act by oxidative demethylation. Epe1 modulates the stability of silent chromatin and contains a JmjC domain. The Epe1 protein can be modelled onto the structure of the 2‐oxoglutarate‐Fe(II)‐dependent dioxygenase, factor inhibiting hypoxia inducible factor (FIH), which is a protein hydroxylase that also contains a JmjC domain. Thus, Epe1 and certain other chromatin‐associated JmjC‐domain proteins may be protein hydroxylases that catalyse a novel histone modification. Another intriguing possibility is that, by hydroxylating the methyl groups, Epe1 and certain other JmjC‐domain proteins may be able to demethylate mono‐, di‐ or trimethylated histones.

The RNA-editing enzyme ADAR1 controls innate immune responses to RNA.

Summary The ADAR RNA-editing enzymes deaminate adenosine bases to inosines in cellular RNAs. Aberrant interferon expression occurs in patients in whom ADAR1 mutations cause Aicardi-Goutières syndrome (AGS) or dystonia arising from striatal neurodegeneration. Adar1 mutant mouse embryos show aberrant interferon induction and die by embryonic day E12.5. We demonstrate that Adar1 embryonic lethality is rescued to live birth in Adar1; Mavs double mutants in which the antiviral interferon induction response to cytoplasmic double-stranded RNA (dsRNA) is prevented. Aberrant immune responses in Adar1 mutant mouse embryo fibroblasts are dramatically reduced by restoring the expression of editing-active cytoplasmic ADARs. We propose that inosine in cellular RNA inhibits antiviral inflammatory and interferon responses by altering RLR interactions. Transfecting dsRNA oligonucleotides containing inosine-uracil base pairs into Adar1 mutant mouse embryo fibroblasts reduces the aberrant innate immune response. ADAR1 mutations causing AGS affect the activity of the interferon-inducible cytoplasmic isoform more severely than the nuclear isoform.

Catalysis in the crystal: synchrotron radiation studies with glycogen phosphorylase b.

Direct observation of the progress of a catalysed reaction in crystals of glycogen phosphorylase b has been made possible through fast crystallographic data collection achieved at the Synchrotron Radiation source at Daresbury, UK. In the best experiments, data to 2.7 A resolution (some 108,300 measurements; 21,200 unique reflections) were measured in 25 min. In a series of time‐resolved studies in which the control properties of the enzyme were exploited in order to slow down the reaction, the conversion of heptenitol to heptulose‐2‐phosphate, the phosphorylysis of maltoheptaose to yield glucose‐1‐phosphate and the oligosaccharide synthesis reaction involving maltotriose and glucose‐1‐phosphate have been monitored in the crystal. Changes in electron density in the difference Fourier maps are observed as the reaction proceeds not only at the catalytic site but also the allosteric and glycogen storage sites. Phosphorylase b is present in the crystals in the T state and under these conditions exhibits low affinity for both phosphate and oligosaccharide substrates. There are pronounced conformational changes associated with the formation and binding of the high‐affinity dead‐end product, heptulose‐2‐phosphate, which show that movement of an arginine residue, Arg 569, is critical for formation of the substrate‐phosphate recognition site. The results are discussed with reference to proposals for the enzymic mechanism of phosphorylase. The feasibility for time‐resolved studies on other systems and recent advances in this area utilizing Laue diffraction are also discussed.

A NASP (N1/N2)-Related Protein, Sim3, Binds CENP-A and Is Required for Its Deposition at Fission Yeast Centromeres

Summary A defining feature of centromeres is the presence of the histone H3 variant CENP-ACnp1. It is not known how CENP-ACnp1 is specifically delivered to, and assembled into, centromeric chromatin. Through a screen for factors involved in kinetochore integrity in fission yeast, we identified Sim3. Sim3 is homologous to known histone binding proteins NASPHuman and N1/N2Xenopus and aligns with Hif1S. cerevisiae, defining the SHNi-TPR family. Sim3 is distributed throughout the nucleoplasm, yet it associates with CENP-ACnp1 and also binds H3. Cells defective in Sim3 function have reduced levels of CENP-ACnp1 at centromeres (and increased H3) and display chromosome segregation defects. Sim3 is required to allow newly synthesized CENP-ACnp1 to accumulate at centromeres in S and G2 phase-arrested cells in a replication-independent mechanism. We propose that one function of Sim3 is to act as an escort that hands off CENP-ACnp1 to chromatin assembly factors, allowing its incorporation into centromeric chromatin.

Gelsolin Domains 4–6 in Active, Actin-free Conformation Identifies Sites of Regulatory Calcium Ions

Structural analysis of gelsolin domains 4-6 demonstrates that the two highest-affinity calcium ions that activate the molecule are in domains 5 and 6, one in each. An additional calcium site in domain 4 depends on subsequent actin binding and is seen only in the complex. The uncomplexed structure is primed to bind actin. Since the disposition of the three domains is similar in different crystal environments, either free or in complex with actin, the conformation in calcium is intrinsic to active gelsolin itself. Thus the actin-free structure shows that the structure with an actin monomer is a good model for an actin filament cap. The last 13 residues of domain 6 have been proposed to be a calcium-activated latch that, in the inhibited form only, links two halves of gelsolin. Comparison with the active structure shows that loosening of the latch contributes but is not central to activation. Calcium binding in domain 6 invokes a cascade of swapped ion-pairs. A basic residue swaps acidic binding partners to stabilise a straightened form of a helix that is kinked in inhibited gelsolin. The other end of the helix is connected by a loop to an edge beta-strand. In active gelsolin, an acidic residue in this helix breaks with its loop partner to form a new intrahelical ion-pairing, resulting in the breakage of the continuous sheet between domains 4 and 6, which is central to the inhibited conformation. A structural alignment of domain sequences provides a rationale to understand why the two calcium sites found here have the highest affinity amongst the five different candidate sites found in other gelsolin structures.

Domain 2 of gelsolin binds directly to tropomyosin.

Gelsolin is an actin filament severing protein composed of six similar structured domains that differ with respect to actin, calcium and polyphospho‐inositide binding. Previous work has established that gelsolin binds tropomyosin [Koepf, E.K. and Burtnick, L.D. (1992) FEBS Lett. 309, 56–58]. We have produced various specific gelsolin domains in Escherichia coli in order to establish which of the six domains binds tropomyosin. Gelsolin domains 1–3 (G1–3), G1–2 and G2 all bind tropomyosin in a pH and calcium insensitive manner whereas binding of G4–6 to tropomyosin was barely detectable under the conditions tested. We conclude that gelsolin binds tropomyosin via domain 2 (G2).