Monique Gangloff

Assembly and localization of Toll-like receptor signalling complexes

Signal transduction by the Toll-like receptors (TLRs) is central to host defence against many pathogenic microorganisms and also underlies a large burden of human disease. Thus, the mechanisms and regulation of signalling by TLRs are of considerable interest. In this Review, we discuss the molecular basis for the recognition of pathogen-associated molecular patterns, the nature of the protein complexes that mediate signalling, and the way in which signals are regulated and integrated at the level of allosteric assembly, post-translational modification and subcellular trafficking of the components of the signalling complexes. These fundamental molecular mechanisms determine whether the signalling output leads to a protective immune response or to serious pathologies such as sepsis. A detailed understanding of these processes at the molecular level provides a rational framework for the development of new drugs that can specifically target pathological rather than protective signalling in inflammatory and autoimmune disease.

The molecular basis of the host response to lipopolysaccharide

Lipopolysaccharide (LPS), which is produced by Gram-negative bacteria, is a powerful activator of innate immune responses. LPS binds to the proteins Toll-like receptor 4 (TLR4) and MD2 to activate pro-inflammatory signalling pathways. The TLR4–MD2 receptor complex is crucial for the host recognition of Gram-negative bacterial infection, and pathogens have devised many strategies to evade or manipulate TLR4–MD2 activity. The TLR4–MD2 signalling pathway is therefore potentially an important therapeutic target. This Progress article focuses on recent exciting data that have revealed the structural basis of TLR4–MD2 recognition of LPS.

Toll-like receptors as molecular switches

Members of the Toll family of single-pass transmembrane receptors are key mediators of innate immunity in both vertebrates and invertebrates. They respond to various pathogen-associated stimuli and transduce the complex signalling responses that are required for inflammation and for the subsequent development of adaptive immunity. Here, we propose a molecular mechanism for signalling by the Toll and Toll-like receptors that involves a series of protein conformational changes initiated by dimerization of their extracellular domains. The initial dimerization event, which is triggered by the interaction of the receptor with its ligand, might disrupt a pre-formed but non-functional dimer. Formation of a stable receptor–ligand complex then relieves constitutive autoinhibition, enabling receptor–receptor association of the extracellular juxtamembrane regions and cytoplasmic signalling domains. This activation process constitutes a tightly regulated, unidirectional molecular switch.

What the Myddosome structure tells us about the initiation of innate immunity

Signaling by the toll-like receptor (TLR) and interleukin-1 receptor superfamily requires the adapter protein myeloid differentiation primary response protein 88 (MyD88). The recent determination of the structure of the so-called Myddosome provides us with new insights into the structural basis for innate immune signaling. Other information on the biochemistry and genetics of MyD88 and other adapters, such as MyDD adapter-like and TRIF-related adapter molecule, allows us to describe in some detail the signaling process activated by TLRs and provides new insights into the role these important proteins play in innate immunity.

Elucidation of the MD-2/TLR4 Interface Required for Signaling by Lipid IVa

LPS signals through a membrane bound-complex of the lipid binding protein MD-2 and the receptor TLR4. In this study we identify discrete regions in both MD-2 and TLR4 that are required for signaling by lipid IVa, an LPS derivative that is an agonist in horse but an antagonist in humans. We show that changes in the electrostatic surface potential of both MD-2 and TLR4 are required in order that lipid IVa can induce signaling. In MD-2, replacing horse residues 57–66 and 82–89 with the equivalent human residues confers a level of constitutive activity on horse MD-2, suggesting that conformational switching in this protein is likely to be important in ligand-induced activation of MD-2/TLR4. We identify leucine-rich repeat 14 in the C terminus of TLR4 as essential for lipid IVa activation of MD-2/TLR4. Remarkably, we identify a single residue in the glycan-free flank of the horse TLR4 solenoid that confers the ability to signal in response to lipid IVa. These results suggest a mechanism of signaling that involves crosslinking mediated by both MD-2-receptor and receptor-receptor contacts in a model that shows striking similarities to the recently published structure (Cell 130: 1071–1082) of the ligand-bound TLR1/2 ectodomain heterodimer.

Estrogen receptor transcription and transactivation: Structure-function relationship in DNA- and ligand-binding domains of estrogen receptors

Estrogen receptors are members of the nuclear receptor steroid family that exhibit specific structural features, ligand-binding domain sequence identity and dimeric interactions, that single them out. The crystal structures of their DNA-binding domains give some insight into how nuclear receptors discriminate between DNA response elements. The various ligand-binding domain crystal structures of the two known estrogen receptor isotypes (α and β) allow one to interpret ligand specificity and reveal the interactions responsible for stabilizing the activation helix H12 in the agonist and antagonist positions.

Ligand-Receptor and Receptor-Receptor Interactions Act in Concert to Activate Signaling in the Drosophila Toll Pathway

In Drosophila, the signaling pathway mediated by the Toll receptor is critical for the establishment of embryonic dorso-ventral pattern and for innate immune responses to bacterial and fungal pathogens. Toll is activated by high affinity binding of the cytokine Spätzle, a dimeric ligand of the cystine knot family. In vertebrates, a related family of Toll-like receptors play a critical role in innate immune responses. Despite the importance of this family of receptors, little is known about the biochemical events that lead to receptor activation and signaling. Here, we show that Spätzle binds to the N-terminal region of Toll and, using biophysical methods, that the binding is complex. The two binding events that cause formation of the cross-linked complex are non-equivalent: the first Toll ectodomain binds Spätzle with an affinity 3-fold higher than the second molecule suggesting that pathway activation involves negative cooperativity. We further show that the Toll ectodomains are able to form low affinity dimers in solution and that juxtamembrane sequences of Toll are critical for the activation or derepression of the pathway. These results, taken together, suggest a mechanism of signal transduction that requires both ligand-receptor and receptor-receptor interactions.

Allergens as Immunomodulatory Proteins: The Cat Dander Protein Fel d 1 Enhances TLR Activation by Lipid Ligands

Allergic responses can be triggered by structurally diverse allergens. Most allergens are proteins, yet extensive research has not revealed how they initiate the allergic response and why the myriad of other inhaled proteins do not. Among these allergens, the cat secretoglobulin protein Fel d 1 is a major allergen and is responsible for severe allergic responses. In this study, we show that similar to the mite dust allergen Der p 2, Fel d 1 substantially enhances signaling through the innate receptors TLR4 and TLR2. In contrast to Der p 2, however, Fel d 1 does not act by mimicking the TLR4 coreceptor MD2 and is not able to bind stably to the TLR4/MD2 complex in vitro. Fel d 1 does, however, bind to the TLR4 agonist LPS, suggesting that a lipid transfer mechanism may be involved in the Fel d 1 enhancement of TLR signaling. We also show that the dog allergen Can f 6, a member of a distinct class of lipocalin allergens, has very similar properties to Fel d 1. We propose that Fel d 1 and Can f 6 belong to a group of allergen immunomodulatory proteins that enhance innate immune signaling and promote airway hypersensitivity reactions in diseases such as asthma.

Structural insight into the mechanism of activation of the toll receptor by the dimeric ligand Spätzle

The Drosophila Toll receptor, which functions in both embryonic patterning and innate immunity to fungi and Gram-positive bacteria, is activated by a dimeric cytokine ligand, Spätzle (Spz). Previous studies have suggested that one Spz cross-links two Toll receptor molecules to form an activated complex. Here we report electron microscopy structures of the Toll ectodomain in the absence and presence of Spz. Contrary to expectations, Spz does not directly cross-link two Toll ectodomains. Instead, Spz binding at the N-terminal end of Toll predominantly induces the formation of a 2:2 complex, with two sites of interaction between the ectodomain chains, one located near to the N terminus of the solenoid and the other between the C-terminal juxtamembrane sequences. Moreover, Toll undergoes a ligand-induced conformational change, becoming more tightly curved than in the apo form. The unexpected 2:2 complex was confirmed by mass spectrometry under native conditions. These results suggest that activation of Toll is an allosteric mechanism induced by an end-on binding mode of its ligand Spz.

Molecular Mechanism That Induces Activation of Spätzle, the Ligand for the Drosophila Toll Receptor

The Drosophila Toll receptor is activated by an endogenous cytokine ligand Spätzle. Active ligand is generated in response to positional cues in embryonic dorso-ventral patterning and microbial pathogens in the insect immune response. Spätzle is secreted as a pro-protein and is processed into an active form by the serine endoproteases Easter and Spätzle-processing enzyme during dorso-ventral patterning and infection, respectively. Here, we provide evidence for the molecular mechanism of this activation process. We show that the Spätzle prodomain masks a predominantly hydrophobic region of Spätzle and that proteolysis causes a conformational change that exposes determinants that are critical for binding to the Toll receptor. We also gather that a conserved sequence motif in the prodomain presents features of an amphipathic helix likely to bind a hydrophobic cleft in Spätzle thereby occluding the putative Toll binding region. This mechanism of activation has a striking similarity to that of coagulogen, a clotting factor of the horseshoe crab, an invertebrate that has changed little in 400 million years. Taken together, our findings demonstrate that an ancient passive defense system has been adapted during evolution and converted for use in a critical pathway of innate immune signaling and embryonic morphogenesis.