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Toll-like receptor (TLR) signaling is a key innate immunity response to pathogens. Recruitment of signaling adapters such as MAL (TIRAP) and MyD88 to the TLRs requires Toll/interleukin-1 receptor (TIR)-domain interactions, which remain structurally elusive. Here we show that MAL TIR domains spontaneously and reversibly form filaments in vitro. They also form cofilaments with TLR4 TIR domains and induce formation of MyD88 assemblies. A 7-Å-resolution cryo-EM structure reveals a stable MAL protofilament consisting of two parallel strands of TIR-domain subunits in a BB-loop-mediated head-to-tail arrangement. Interface residues that are important for the interaction are conserved among different TIR domains. Although large filaments of TLR4, MAL or MyD88 are unlikely to form during cellular signaling, structure-guided mutagenesis, combined with in vivo interaction assays, demonstrated that the MAL interactions defined within the filament represent a template for a conserved mode of TIR-domain interaction involved in both TLR and interleukin-1 receptor signaling.

MyD88 adaptor-like (MAL) is a critical protein in innate immunity, involved in signaling by several Toll-like receptors (TLRs), key pattern recognition receptors (PRRs). Crystal structures of MAL revealed a nontypical Toll/interleukin-1 receptor (TIR)-domain fold stabilized by two disulfide bridges. We therefore undertook a structural and functional analysis of the role of reactive cysteine residues in the protein. Under reducing conditions, the cysteines do not form disulfides, but under oxidizing conditions they are highly amenable to modification. The solution structure of the reduced form of the MAL TIR domain, determined by NMR spectroscopy, reveals a remarkable structural rearrangement compared with the disulfide-bonded structure, which includes the relocation of a β-strand and repositioning of the functionally important "BB-loop" region to a location more typical for TIR domains. Redox measurements by NMR further reveal that C91 has the highest redox potential of all cysteines in MAL. Indeed, mass spectrometry revealed that C91 undergoes glutathionylation in macrophages activated with the TLR4 ligand lipopolysaccharide (LPS). The C91A mutation limits MAL glutathionylation and acts as a dominant negative, blocking the interaction of MAL with its downstream target MyD88. The H92P mutation mimics the dominant-negative effects of the C91A mutation, presumably by preventing C91 glutathionylation. The MAL C91A and H92P mutants also display diminished degradation and interaction with interleukin-1 receptor-associated kinase 4 (IRAK4). We conclude that in the cell, MAL is not disulfide-bonded and requires glutathionylation of C91 for signaling.

The activation of the Toll-like receptors (TLRs) on eukaryotic cell membranes results in the cytosolicTIR domains coming into close proximity for the binding of adaptor proteins to form a scaffold fordownstream signalling. MAL/TIRAP (MyD88 (myeloid differentiation primary response gene 88)adaptor-like/TIR domain-containing adaptor protein) is the bridging adaptor required for MyD88-dependent signaling by a number of TLRs including TLR4. Crystal structures of the MAL TIR domain(MALTIR) have shown that the structure of the TIR domain is strikingly different to a typical TIRdomain fold. The MALTIR crystal structure contains a long AB loop situated between the aA helix andthe bB strand, but no BB loop. This structural re-arrangement has significance as the BB loop in otherTIR domains has been associated with protein interactions. In addition, four of the seven cysteineresidues were oxidised in the crystal structures, participating in two disulfide bonds. To understandthe role of these cysteines, comprehensive biochemical tests and structural characterisation of theprotein was carried out.This thesis presents an NMR solution structure of the MALTIRC116A mutant, which shows a significantstructural rearrangement compared to the crystal structures. The solution structure of MALTIRC116Apresented in this thesis shows a typical TIR domain fold with the functionally important long BBloop.Strikingly, all seven cysteine residues of this structure appear to be reduced and do not formdisulfide bonds. This was not surprising given that the cytosol in which the MAL protein resides ishighly reducing. In addition, all cysteines are amendable to modification, such as glutathionylation,and have varying sensitivities in physiological redox conditions. Of particular note, the peak intensityof cysteine 91 decreased by 45% when the redox environment was changed from a physiologicallyreducing -225 mV to an oxidising -190 mV, suggesting that this cysteine may play an importantfunctional role.Further to this, the data presented here shows that the MALTIR resides in a dynamic equilibrium thatcan be shifted between a monomeric and oligomeric state when exposed to different pH, salt,temperature, and concentration. The identification of this reversible oligomerisation is remarkableand provides a possible mechanism for the regulation of this signalling pathway. Followingpurification, a dimer species can also separated. This species is not in equilibrium with the monomerand is not covalently bound to the monomer.Similarly to TIR domains, coiled-coil (CC) domains in plants are involved in defence signalling andimplicated in homotypic interactions. In this thesis, the NMR solution structure of a fragment,consisting of residues 6n120, of the wheat steam rust protein Sr33 CC domain is presented. The structure corresponds to a four-helix bundle with a surface-exposed functionally important EDVIDmotif. This is significantly different to the published crystal structure of the closely related CCdomain from the orthologous barley immunity receptor MLA10 that confers resistance to powderymildew; however, the structures share striking similarity to the more distantly related Rx protein CCdomain from potato. Biophysical studies demonstrate that in solution, all three structures aremonomeric and adopt a similar fold. Further to this, data shows that the minimal functional unitconsists of residues 1n142 and that in planta, self-association correlates strongly with immunityinducedcell death. Collectively, this work contributes to the structural and functional understandingof the CC domains in plants.

NMR spectroscopy is a powerful method in structural and functional analysis of macromolecules and has become particularly prevalent in studies of protein structure, function and dynamics. Unique to NMR spectroscopy is the relatively low constraints on sample preparation and the high level of control of sample conditions. Proteins can be studied in a wide range of buffer conditions, e.g. different pHs and variable temperatures, allowing studies of proteins under conditions that are closer to their native environment compared to other structural methods such as X-ray crystallography and electron microscopy. The key disadvantage of NMR is the relatively low sensitivity of the method, requiring either concentrated samples or very lengthy data-acquisition times. Thus, proteins that are unstable or can only be studied in dilute solutions are often considered practically unfeasible for NMR studies. Here, we describe a general method, where non-uniform sampling (NUS) allows for signal averaging to be monitored in an iterative manner, enabling efficient use of spectrometer time, ultimately leading to savings in costs associated with instrument and isotope-labelled protein use. The method requires preparation of multiple aliquots of the protein sample that are flash-frozen and thawed just before acquisition of a short NMR experiments carried out while the protein is stable (12 h in the presented case). Non-uniform sampling enables sufficient resolution to be acquired for each short experiment. Identical NMR datasets are acquired and sensitivity is monitored after each co-added spectrum is reconstructed. The procedure is repeated until sufficient signal-to-noise is obtained. We discuss how maximum entropy reconstruction is used to process the data, and propose a variation on the previously described method of automated parameter selection. We conclude that combining NUS with iterative co-addition is a general approach, and particularly powerful when applied to unstable proteins.

Plants use intracellular immunity receptors, known as nucleotide-binding oligomerization domain-like receptors (NLRs), to recognize specific pathogen effector proteins and induce immune responses. These proteins provide resistance to many of the world’s most destructive plant pathogens, yet we have a limited understanding of the molecular mechanisms that lead to defense signaling. We examined the wheat NLR protein, Sr33, which is responsible for strain-specific resistance to the wheat stem rust pathogen, Puccinia graminis f. sp. tritici. We present the solution structure of a coiled-coil (CC) fragment from Sr33, which adopts a four-helix bundle conformation. Unexpectedly, this structure differs from the published dimeric crystal structure of the equivalent region from the orthologous barley powdery mildew resistance protein, MLA10, but is similar to the structure of the distantly related potato NLR protein, Rx. We demonstrate that these regions are, in fact, largely monomeric and adopt similar folds in solution in all three proteins, suggesting that the CC domains from plant NLRs adopt a conserved fold. However, larger C-terminal fragments of Sr33 and MLA10 can self-associate both in vitro and in planta, and this self-association correlates with their cell death signaling activity. The minimal region of the CC domain required for both cell death signaling and self-association extends to amino acid 142, thus including 22 residues absent from previous biochemical and structural protein studies. These data suggest that self-association of the minimal CC domain is necessary for signaling but is likely to involve a different structural basis than previously suggested by the MLA10 crystallographic dimer.

Motility helps many pathogens swim through the highly viscous intestinal mucus. Given the differing outcomes of Campylobacter concisus infection, the motility of eight C. concisus strains isolated from patients with Crohn’s disease (n=3), acute (n=3) and chronic (n=1) gastroenteritis and a healthy control (n=1) were compared. Following growth on solid or liquid media the eight strains formed two groups; however, the type of growth medium did not affect motility. In contrast, following growth in viscous liquid medium seven of the eight strains demonstrated significantly decreased motility. In media of increasing viscosities the motility of C. concisus UNSWCD had two marked increases at viscosities of 20.0 and 74.7 centipoises. Determination of the ability of UNSWCD to swim through a viscous medium, adhere to and invade intestinal epithelial cells showed that while adherence levels significantly decreased with increasing viscosity, invasion levels did not significantly change. In contrast, adherence to and invasion of UNSWCD to mucus-producing intestinal cells increased upon accumulation of mucus, as did bacterial aggregation. Given this aggregation, we determined the ability of the eight C. concisus strains to form biofilms, and showed that all strains formed biofilms. In conclusion, the finding that C. concisus strains could be differentiated into two groups based on their motility may suggest that strains with high motility have an increased ability to swim through the intestinal mucus and reach the epithelial layer.