Andrew M. Ellisdon

A Common Fold Mediates Vertebrate Defense and Bacterial Attack

Proteins containing membrane attack complex/perforin (MACPF) domains play important roles in vertebrate immunity, embryonic development, and neural-cell migration. In vertebrates, the ninth component of complement and perforin form oligomeric pores that lyse bacteria and kill virus-infected cells, respectively. However, the mechanism of MACPF function is unknown. We determined the crystal structure of a bacterial MACPF protein, Plu-MACPF from Photorhabdus luminescens, to 2.0 angstrom resolution. The MACPF domain reveals structural similarity with poreforming cholesterol-dependent cytolysins (CDCs) from Gram-positive bacteria. This suggests that lytic MACPF proteins may use a CDC-like mechanism to form pores and disrupt cell membranes. Sequence similarity between bacterial and vertebrate MACPF domains suggests that the fold of the CDCs, a family of proteins important for bacterial pathogenesis, is probably used by vertebrates for defense against infection.

The two-stage pathway of ataxin-3 Fibrillogenesis involves a polyglutamine-independent step

The aggregation of ataxin-3 is associated with spinocerebellar ataxia type 3, which is characterized by the formation of intraneuronal aggregates. However, the mechanism of aggregation is currently not well understood. Ataxin-3 consists of a folded Josephin domain followed by two ubiquitin-interacting motifs and a C-terminal polyglutamine tract, which in the non-pathological form is less than 45 residues in length. We demonstrate that ataxin-3 with 64 glutamines (at(Q64)) undergoes a two-stage aggregation. The first stage involves formation of SDS-soluble aggregates, and the second stage results in formation of SDS-insoluble aggregates via the poly(Q) region. Both these first and second stage aggregates display typical amyloid-like characteristics. Under the same conditions at(Q15) and at(QHQ) undergo a single step aggregation event resulting in SDS-soluble aggregates, which does not involve the polyglutamine tract. These aggregates do not convert to the SDS-insoluble form. These observations demonstrate that ataxin-3 has an inherent capacity to aggregate through its non-polyglutamine domains. However, the presence of a pathological length polyglutamine tract introduces an additional step resulting in formation of a highly stable amyloid-like aggregate.

IL-37 requires the receptors IL-18Rα and IL-1R8 (SIGIRR) to carry out its multifaceted anti-inflammatory program upon innate signal transduction

Interleukin 37 (IL-37) and IL-1R8 (SIGIRR or TIR8) are anti-inflammatory orphan members of the IL-1 ligand family and IL-1 receptor family, respectively. Here we demonstrate formation and function of the endogenous ligand-receptor complex IL-37–IL-1R8–IL-18Rα. The tripartite complex assembled rapidly on the surface of peripheral blood mononuclear cells upon stimulation with lipopolysaccharide. Silencing of IL-1R8 or IL-18Rα impaired the anti-inflammatory activity of IL-37. Whereas mice with transgenic expression of IL-37 (IL-37tg mice) with intact IL-1R8 were protected from endotoxemia, IL-1R8-deficient IL-37tg mice were not. Proteomic and transcriptomic investigations revealed that IL-37 used IL-1R8 to harness the anti-inflammatory properties of the signaling molecules Mer, PTEN, STAT3 and p62(dok) and to inhibit the kinases Fyn and TAK1 and the transcription factor NF-κB, as well as mitogen-activated protein kinases. Furthermore, IL-37–IL-1R8 exerted a pseudo-starvational effect on the metabolic checkpoint kinase mTOR. IL-37 thus bound to IL-18Rα and exploited IL-1R8 to activate a multifaceted intracellular anti-inflammatory program.

Sus1, Cdc31, and the Sac3 CID region form a conserved interaction platform that promotes nuclear pore association and mRNA export.

Summary The yeast Sac3:Cdc31:Sus1:Thp1 (TREX-2) complex facilitates the repositioning and association of actively transcribing genes with nuclear pores (NPCs)—“gene gating”—that is central to integrating transcription, processing, and mRNA nuclear export. We present here the crystal structure of Sus1 and Cdc31 bound to a central region of Sac3 (the CID domain) that is crucial for its function. Sac3CID forms a long, gently undulating α helix around which one Cdc31 and two Sus1 chains are wrapped. Sus1 has an articulated helical hairpin fold that facilitates its wrapping around Sac3. In vivo studies using engineered mutations that selectively disrupted binding of individual chains to Sac3 indicated that Sus1 and Cdc31 function synergistically to promote NPC association of TREX-2 and mRNA nuclear export. These data indicate Sac3CID provides a scaffold within TREX-2 to integrate interactions between protein complexes to facilitate the coupling of transcription and mRNA export during gene expression.

Human embryonic stem cell models of Huntington disease

Huntington disease (HD) is an incurable late-onset neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the HD gene (HTT). The major hallmark of disease pathology is neurodegeneration in the brain. Currently, there are no useful in-vitro human models of HD. Recently, two human embryonic stem cell (hESC) lines carrying partial (CAG(37)) and fully (CAG(51)) penetrant mutant alleles have been derived from affected IVF embryos identified following preimplantation genetic diagnosis (PGD). Fluorescence polymerase chain reaction (F-PCR) and Genescan analysis confirmed the original embryonic HD genotypes. Reverse transcription PCR (RT-PCR) analysis confirmed the expression of mutant transcripts and western blot analysis demonstrated expression of mutant huntingtin protein (HTT). After treatment with noggin, HD hESC formed neurospheres, which could be further differentiated into cells susceptible to neurodegeneration in HD, namely primary neurones and astrocytes. Small pool PCR analysis of neurosphere cells revealed instability of disease-length CAG repeats following differentiation. The presence of active HTT genes, neural differentiation capabilities and evidence of CAG repeat instability indicates these HD hESC lines may serve as valuable in-vitro human models of HD to better understand the mechanisms of neurodegeneration in patients, and for drug screening to identify new therapies for human clinical trials.

Structural basis for the assembly and nucleic acid binding of the TREX-2 transcription-export complex.

The conserved TREX-2 transcription-export complex integrates transcription and processing of many actively transcribed nascent mRNAs with the recruitment of export factors at nuclear pores and also contributes to transcriptional memory and genomic stability. We report the crystal structure of the Sac3–Thp1–Sem1 segment of Saccharomyces cerevisiae TREX-2 that interfaces with the gene expression machinery. Sac3–Thp1–Sem1 forms a previously uncharacterized PCI-domain complex characterized by the juxtaposition of Sac3 and Thp1 winged helix domains, forming a platform that mediates nucleic acid binding. Our structure-guided mutations support the idea that the Thp1-Sac3 interaction is an essential requirement for mRNA binding and for the coupling of transcription and processing to mRNP assembly and export. These results provide insight into how newly synthesized transcripts are efficiently transferred from TREX-2 to the principal mRNA export factor, and they reveal how Sem1 stabilizes PCI domain–containing proteins and promotes complex assembly.

The Structural Impact of a Polyglutamine Tract Is Location-Dependent

Polyglutamine (polyQ) expansion leads to protein aggregation and neurodegeneration in Huntingtons disease and eight other inherited neurological conditions. Expansion of the polyQ tract beyond a threshold of 37 glutamines leads to the formation of toxic nuclear aggregates. This suggests that polyQ expansion causes a conformational change within the protein, the nature of which is unclear. There is a trend in the disease proteins that the polyQ tract is located external to but not within a structured domain. We have created a model polyQ protein in which the repeat location mimics the flexible environment of the polyQ tract in the disease proteins. Our model protein recapitulates the aggregation features observed with the clinical proteins and allows structural characterization. With the use of NMR spectroscopy and a range of biophysical techniques, we demonstrate that polyQ expansion into the pathological range has no effect on the structure, dynamics, and stability of a domain adjacent to the polyQ tract. To explore the clinical significance of repeat location, we engineered a variant of the model protein with a polyQ tract within the domain, a location that does not mimic physiological context, demonstrating significant destabilization and structural perturbation. These different effects highlight the importance of repeat location. We conclude that protein misfolding within the polyQ tract itself is the driving force behind the key characteristics of polyQ disease, and that structural perturbation of flanking domains is not required.

Structural basis for the interaction between yeast Spt-Ada-Gcn5 acetyltransferase (SAGA) complex components Sgf11 and Sus1.

Sus1 is a central component of the yeast gene gating machinery, the process by which actively transcribing genes such as GAL1 become associated with nuclear pore complexes. Sus1 is a component of both the SAGA transcriptional co-activator complex and the TREX-2 complex that binds to nuclear pore complexes. TREX-2 contains two Sus1 chains that have an articulated helical hairpin fold, enabling them to wrap around an extended α-helix in Sac3, following a helical hydrophobic stripe. In SAGA, Sus1 binds to Sgf11 and has been proposed to provide a link between SAGA and TREX-2. We present here the crystal structure of the complex between Sus1 and the N-terminal region of Sgf11 that forms an extended α-helix around which Sus1 wraps in a manner that shares some similarities with the Sus1-Sac3 interface in TREX-2. However, the Sus1-binding site on Sgf11 is somewhat shorter than on Sac3 and is based on a narrower hydrophobic stripe. Engineered mutants that disrupt the Sgf11-Sus1 interaction in vitro confirm the importance of the hydrophobic helical stripe in molecular recognition. Helix α1 of the Sus1-articulated hairpin does not bind directly to Sgf11 and adopts a wide range of conformations within and between crystal forms, consistent with the presence of a flexible hinge and also with results from previous extensive mutagenesis studies (Klöckner, C., Schneider, M., Lutz, S., Jani, D., Kressler, D., Stewart, M., Hurt, E., and Köhler, A. (2009) J. Biol. Chem. 284, 12049–12056). A single Sus1 molecule cannot bind Sgf11 and Sac3 simultaneously and this, combined with the structure of the Sus1-Sgf11 complex, indicates that Sus1 forms separate subcomplexes within SAGA and TREX-2.

Structural biology of the PCI-protein fold.

The PCI fold is based on a stack of α-helices topped with a winged-helix domain and is found in a range of proteins that form central parts of large complexes such as the proteasome lid, the COP9 signalosome, elongation factor eIF3, and the TREX-2 complex. Recent structural determinations have given intriguing insight into how these folds function both to facilitate the generation of larger proteinaceous assembles and also to interact functionally with nucleic acids.

The Phosphatidylinositol (3,4,5)-trisphosphate-dependent Rac Exchanger 1:Ras-related C3 Botulinum Toxin Substrate 1 (P-Rex1:Rac1) Complex Reveals the Basis of Rac1 Activation in Breast Cancer Cells

Background: P-Rex1 (phosphatidylinositol (3,4,5)-trisphosphate-dependent Rac exchanger-1) activity is correlated with tumorigenesis in cancer. Results: P-Rex1·Rac1 (Ras-related C3 botulinum toxin substrate-1) crystal structure reveals the molecular mechanism of Rac1 activation. Conclusion: The P-Rex1·Rac1 interface is critical for Rac1 activation in breast cancer cell lines. Significance: The study provides a rationale for therapeutic targeting of the P-Rex1·Rac1 interface by describing the structural basis of P-Rex1 activity. The P-Rex (phosphatidylinositol (3,4,5)-trisphosphate (PIP3)-dependent Rac exchanger) family (P-Rex1 and P-Rex2) of the Rho guanine nucleotide exchange factors (Rho GEFs) activate Rac GTPases to regulate cell migration, invasion, and metastasis in several human cancers. The family is unique among Rho GEFs, as their activity is regulated by the synergistic binding of PIP3 and Gβγ at the plasma membrane. However, the molecular mechanism of this family of multi-domain proteins remains unclear. We report the 1.95 Å crystal structure of the catalytic P-Rex1 DH-PH tandem domain in complex with its cognate GTPase, Rac1 (Ras-related C3 botulinum toxin substrate-1). Mutations in the P-Rex1·Rac1 interface revealed a critical role for this complex in signaling downstream of receptor tyrosine kinases and G protein-coupled receptors. The structural data indicated that the PIP3/Gβγ binding sites are on the opposite surface and markedly removed from the Rac1 interface, supporting a model whereby P-Rex1 binding to PIP3 and/or Gβγ releases inhibitory C-terminal domains to expose the Rac1 binding site.