Signalling by co-operative higher-order assembly formation: linking evidence at molecular and cellular levels.

The concept of higher-order assembly signalling or signalling by co-operative assembly formation (SCAF) was proposed based on the structures of signalling assemblies formed by proteins featuring domains from the death-fold family and the Toll/interleukin-1 receptor domain family. Because these domains form filamentous assemblies upon stimulation and activate downstream pathways through induced proximity, they were envisioned to sharpen response thresholds through the extreme co-operativity of higher-order assembly. Recent findings demonstrate that a central feature of the SCAF mechanism is the nucleation barrier that allows a switch-like, digital or 'all-or-none' response to minute stimuli.

Employs a Rare Fatty Acid Desaturase for Niche-Specific Host Adaptation.

is a hospital-associated pathogen with unique fatty acid homeostasis features. This includes a reliance on desaturases for proliferation, due to an inability to generate unsaturated fatty acids during the synthesis cycles. However, there are various unexplained gaps in fatty acid homeostasis, such as the desaturation of synthesized fatty acids.

Structural basis for TIR domain-mediated innate immune signaling by Toll-like receptor adaptors TRIF and TRAM.

Innate immunity relies on Toll-like receptors (TLRs) to detect pathogen-associated molecular patterns. The TIR (Toll/interleukin-1 receptor) domain-containing TLR adaptors TRIF (TIR domain-containing adaptor-inducing interferon-β) and TRAM (TRIF-related adaptor molecule) are essential for MyD88-independent TLR signaling. However, the structural basis of TRIF and TRAM TIR domain-based signaling remains unclear.

Flow cytometric reporter assays provide robust functional analysis of signaling complexes.

Conventional assays to probe signaling protein interactions and function involve measurement of luciferase reporter expression within the bulk cell population, with lack of control over target-protein expression level. To address this issue, we have developed a rapid and robust flow cytometric assay for analysis of signaling protein function. A fluorescent reporter and fluorescent tagging of the target protein enables simultaneous assessment of protein expression and signaling within individual cells.

Cyclic ADP ribose isomers: Production, chemical structures, and immune signaling.

Cyclic adenosine diphosphate (ADP)-ribose (cADPR) isomers are signaling molecules produced by bacterial and plant Toll/interleukin-1 receptor (TIR) domains via nicotinamide adenine dinucleotide (oxidized form) (NAD) hydrolysis. We show that v-cADPR (2'cADPR) and v2-cADPR (3'cADPR) isomers are cyclized by O-glycosidic bond formation between the ribose moieties in ADPR. Structures of 2'cADPR-producing TIR domains reveal conformational changes that lead to an active assembly that resembles those of Toll-like receptor adaptor TIR domains.

The transmembrane adapter SCIMP recruits tyrosine kinase Syk to phosphorylate Toll-like receptors to mediate selective inflammatory outputs.

Innate immune signaling by Toll-like receptors (TLRs) involves receptor phosphorylation, which helps to shape and drive key inflammatory outputs, yet our understanding of the kinases and mechanisms that mediate TLR phosphorylation is incomplete. Spleen tyrosine kinase (Syk) is a nonreceptor protein tyrosine kinase, which is known to relay adaptive and innate immune signaling, including from TLRs. However, TLRs do not contain the conserved dual immunoreceptor tyrosine-based activation motifs that typically recruit Syk to many other receptors.

Crystal structure of the Toll/interleukin-1 receptor (TIR) domain of IL-1R10 provides structural insights into TIR domain signalling.

The Toll/interleukin-1 receptor (TIR) domains are key innate immune signalling modules. Here, we present the crystal structure of the TIR domain of human interleukin-1 receptor 10 (IL-1R10), also called interleukin 1 receptor accessory protein like 2. It is similar to that of IL-1R9 (IL-1RAPL1) but shows significant structural differences to those from Toll-like receptors (TLRs) and the adaptor proteins MyD88 adaptor-like protein (MAL) and MyD88.

Structural Evolution of TIR-Domain Signalosomes.

TIR (Toll/interleukin-1 receptor/resistance protein) domains are cytoplasmic domains widely found in animals and plants, where they are essential components of the innate immune system. A key feature of TIR-domain function in signaling is weak and transient self-association and association with other TIR domains. An additional new role of TIR domains as catalytic enzymes has been established with the recent discovery of NAD-nucleosidase activity by several TIR domains, mostly involved in cell-death pathways.

Crystal structure determination of the armadillo repeat domain of Drosophila SARM1 using MIRAS phasing.

The crystal structure determination of the armadillo repeat motif (ARM) domain of Drosophila SARM1 (dSARM1) is described, which required the combination of a number of sources of phase information in order to obtain interpretable electron-density maps. SARM1 is a central executioner of programmed axon degeneration, a common feature of the early phase of many neurodegenerative diseases. SARM1 is held in the inactive state in healthy axons by its N-terminal auto-inhibitory ARM domain, and is activated to cleave NAD upon injury, triggering subsequent axon degeneration.

MyD88 TIR domain higher-order assembly interactions revealed by microcrystal electron diffraction and serial femtosecond crystallography.

MyD88 and MAL are Toll-like receptor (TLR) adaptors that signal to induce pro-inflammatory cytokine production. We previously observed that the TIR domain of MAL (MAL) forms filaments in vitro and induces formation of crystalline higher-order assemblies of the MyD88 TIR domain (MyD88). These crystals are too small for conventional X-ray crystallography, but are ideally suited to structure determination by microcrystal electron diffraction (MicroED) and serial femtosecond crystallography (SFX).

SARM1 is a metabolic sensor activated by an increased NMN/NAD ratio to trigger axon degeneration.

Axon degeneration is a central pathological feature of many neurodegenerative diseases. Sterile alpha and Toll/interleukin-1 receptor motif-containing 1 (SARM1) is a nicotinamide adenine dinucleotide (NAD)-cleaving enzyme whose activation triggers axon destruction. Loss of the biosynthetic enzyme NMNAT2, which converts nicotinamide mononucleotide (NMN) to NAD, activates SARM1 via an unknown mechanism.

Structural basis for nuclear import selectivity of pioneer transcription factor SOX2.

SOX (SRY-related HMG-box) transcription factors perform critical functions in development and cell differentiation. These roles depend on precise nuclear trafficking, with mutations in the nuclear targeting regions causing developmental diseases and a range of cancers. SOX protein nuclear localization is proposed to be mediated by two nuclear localization signals (NLSs) positioned within the extremities of the DNA-binding HMG-box domain and, although mutations within either cause disease, the mechanistic basis has remained unclear.

Structure, function, and regulation of thioesterases.

Thioesterases are present in all living cells and perform a wide range of important biological functions by catalysing the cleavage of thioester bonds present in a diverse array of cellular substrates. Thioesterases are organised into 25 families based on their sequence conservation, tertiary and quaternary structure, active site configuration, and substrate specificity. Recent structural and functional characterisation of thioesterases has led to significant changes in our understanding of the regulatory mechanisms that govern enzyme activity and their respective cellular roles.

Structural characterization of a short-chain dehydrogenase/reductase from multi-drug resistant Acinetobacter baumannii.

Acinetobacter baumannii (A. baumannii) is a clinically relevant, highly drug-resistant pathogen of global concern. An attractive approach to drug design is to specifically target the type II fatty acid synthesis (FASII) pathway which is critical in Gram negative bacteria and is significantly different to the type I fatty acid synthesis (FASI) pathway found in mammals.

NAD cleavage activity by animal and plant TIR domains in cell death pathways.

SARM1 (sterile alpha and TIR motif containing 1) is responsible for depletion of nicotinamide adenine dinucleotide in its oxidized form (NAD) during Wallerian degeneration associated with neuropathies. Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors recognize pathogen effector proteins and trigger localized cell death to restrict pathogen infection. Both processes depend on closely related Toll/interleukin-1 receptor (TIR) domains in these proteins, which, as we show, feature self-association-dependent NAD cleavage activity associated with cell death signaling.

Structural characterization of β-ketoacyl ACP synthase I bound to platencin and fragment screening molecules at two substrate binding sites.

The bacterial fatty acid pathway is essential for membrane synthesis and a range of other metabolic and cellular functions. The β-ketoacyl-ACP synthases carry out the initial elongation reaction of this pathway, utilizing acetyl-CoA as a primer to elongate malonyl-ACP by two carbons, and subsequent elongation of the fatty acyl-ACP substrate by two carbons. Here we describe the structures of the β-ketoacyl-ACP synthase I from Brucella melitensis in complex with platencin, 7-hydroxycoumarin, and (5-thiophen-2-ylisoxazol-3-yl)methanol.

Death, TIR, and RHIM: Self-assembling domains involved in innate immunity and cell-death signaling.

The innate immune system consists of pattern recognition receptors (PRRs) that detect pathogen- and endogenous danger-associated molecular patterns (PAMPs and DAMPs), initiating signaling pathways that lead to the induction of cytokine expression, processing of pro-inflammatory cytokines, and induction of cell-death responses. An emerging concept in these pathways and associated processes is signaling by cooperative assembly formation (SCAF), which involves formation of higher order oligomeric complexes, and enables rapid and strongly amplified signaling responses to minute amounts of stimulus. Many of these signalosomes assemble through homotypic interactions of members of the death-fold (DF) superfamily, Toll/IL-1 receptor (TIR) domains, or the RIP homotypic interaction motifs (RHIM).

Regulation of signaling by cooperative assembly formation in mammalian innate immunity signalosomes by molecular mimics.

Innate immunity pathways constitute the first line of defense against infections and cellular damage. An emerging concept in these pathways is that signaling involves the formation of finite (e.g.

Structural characterisation of the fatty acid biosynthesis enzyme FabF from the pathogen Listeria monocytogenes.

Development of new antimicrobial agents is required against the causative agent for listeriosis, Listeria monocytogenes, as the number of drug resistant strains continues to increase. A promising target is the β-ketoacyl-acyl carrier protein synthase FabF, which participates in the catalysis of fatty acid synthesis and elongation, and is required for the production of phospholipid membranes, lipoproteins, and lipopolysaccharides. In this study, we report the 1.

Structural Characterisation of FabG from Yersinia pestis, a Key Component of Bacterial Fatty Acid Synthesis.

Ketoacyl-acyl carrier protein reductases (FabG) are ubiquitously expressed enzymes that catalyse the reduction of acyl carrier protein (ACP) linked thioesters within the bacterial type II fatty acid synthesis (FASII) pathway. The products of these enzymes, saturated and unsaturated fatty acids, are essential components of the bacterial cell envelope. The FASII reductase enoyl-ACP reductase (FabI) has been the focus of numerous drug discovery efforts, some of which have led to clinical trials, yet few studies have focused on FabG.

Structural Characterisation of the Beta-Ketoacyl-Acyl Carrier Protein Synthases, FabF and FabH, of Yersinia pestis.

Yersinia pestis, the causative agent of bubonic, pneumonic, and septicaemic plague, remains a major public health threat, with outbreaks of disease occurring in China, Madagascar, and Peru in the last five years. The existence of multidrug resistant Y. pestis and the potential of this bacterium as a bioterrorism agent illustrates the need for new antimicrobials.

Crystallization and preliminary X-ray diffraction analysis of FabG from Yersinia pestis.

The type II fatty-acid biosynthesis pathway of bacteria provides enormous potential for antibacterial drug development owing to the structural differences between this and the type I fatty-acid biosynthesis system found in mammals. β-Ketoacyl-ACP reductase (FabG) is responsible for the reduction of the β-ketoacyl group linked to acyl carrier protein (ACP), and is essential for the formation of fatty acids and bacterial survival. Here, the cloning, expression, purification, crystallization and diffraction of FabG from Yersinia pestis (ypFabG), the highly virulent causative agent of plague, are reported.