Crystallization, X-ray diffraction analysis and preliminary structure determination of the TIR domain from the flax resistance protein L6

The Toll/interleukin-1 receptor (TIR) domain is a protein-protein interaction domain that is found in both animal and plant immune receptors. In animal Toll-like receptor signalling, both homotypic TIR-domain interactions between two receptor molecules and heterotypic interactions between receptors and TIR-domain-containing adaptors are required for initiation of an innate immune response. The TIR domains in cytoplasmic nucleotide-binding/leucine-rich repeat (NB-LRR) plant disease-resistance proteins are not as well characterized, but recent studies have suggested a role in defence signalling. In this study, the crystallization, X-ray diffraction analysis and preliminary structure determination of the TIR domain from the flax resistance protein L6 (L6TIR) are reported. Plate-like crystals of L6TIR were obtained using PEG 200 as a precipitant and diffracted X-rays to 2.3 angstrom resolution. Pseudo-translation complicated the initial assignment of the crystal symmetry, which was ultimately found to correspond to space group P2(1)2(1)2 with two molecules per asymmetric unit. The structure of L6TIR was solved by molecular replacement using the structure of the TIR-domain-containing protein AT1G72930 from Arabidopsis as a template

Long-term behavioural rewriting of maladaptive drinking memories via reconsolidation-update mechanisms

BACKGROUND: Alcohol use disorders can be conceptualised as a learned pattern of maladaptive alcohol-consumption behaviours. The memories encoding these behaviours centrally contribute to long-term excessive alcohol consumption and are therefore an important therapeutic target. The transient period of memory instability sparked during memory reconsolidation offers a therapeutic window to directly rewrite these memories using targeted behavioural interventions. However, clinically-relevant demonstrations of the efficacy of this approach are few. We examined key retrieval parameters for destabilising naturalistic drinking memories and the ability of subsequent counterconditioning to effect long-term reductions in drinking. METHODS: Hazardous/harmful beer-drinking volunteers (N = 120) were factorially randomised to retrieve (RET) or not retrieve (No RET) alcohol reward memories with (PE) or without (No PE) alcohol reward prediction error. All participants subsequently underwent disgust-based counterconditioning of drinking cues. Acute responses to alcohol were assessed pre- and post-manipulation and drinking levels were assessed up to 9 months. RESULTS: Greater long-term reductions in drinking were found when counterconditioning was conducted following retrieval (with and without PE), despite a lack of short-term group differences in motivational responding to acute alcohol. Large variability in acute levels of learning during counterconditioning was noted. 'Responsiveness' to counterconditioning predicted subsequent responses to acute alcohol in RET + PE only, consistent with reconsolidation-update mechanisms. CONCLUSIONS: The longevity of behavioural interventions designed to reduce problematic drinking levels may be enhanced by leveraging reconsolidation-update mechanisms to rewrite maladaptive memory. However, inter-individual variability in levels of corrective learning is likely to determine the efficacy of reconsolidation-updating interventions and should be considered when designing and assessing interventions

Towards the structure of the TIR-domain signalosome

TIR (Toll/interleukin-1 receptor/resistance protein) domains feature in animal, plant and bacterial proteins involved in innate immunity pathways and associated processes. They function through protein:protein interactions, in particular self-association and homotypic association with other TIR domains. Structures of TIR domains from all phyla have been determined, but common association modes have only emerged for plant and bacterial TIR domains, and not for mammalian TIR domains. Numerous attempts involving hybrid approaches, which have combined structural, computational, mutagenesis and biophysical data, have failed to converge onto common models of how these domains associate and function. We propose that the available data can be reconciled in the context of higher-order assembly formation, and that TIR domains function through signaling by cooperative assembly formation (SCAF).The work in the authors’ laboratories was supported by the National Health and Medical Research Council (NHMRC grants 1003326, 1107804, 1071659) and the Australian Research Council (ARC Discovery Projects DP120100685, DP160102244). BK is NHMRC Principal Research Fellow (1003325, 1110971). Simon Williams is funded by ARC DECRA (DE160100893). We acknowledge the use of the University of Queensland Remote Operation Crystallization and X-ray Diffraction Facility (UQ ROCX) and the Australian Synchrotron (MX and SAXSWAXS beamlines) for our structural work

Towards the structure of the TIR-domain signalosome

TIR (Toll/interleukin-1 receptor/resistance protein) domains feature in animal, plant and bacterial proteins involved in innate immunity pathways and associated processes. They function through protein:protein interactions, in particular self-association and homotypic association with other TIR domains. Structures of TIR domains from all phyla have been determined, but common association modes have only emerged for plant and bacterial TIR domains, and not for mammalian TIR domains. Numerous attempts involving hybrid approaches, which have combined structural, computational, mutagenesis and biophysical data, have failed to converge onto common models of how these domains associate and function. We propose that the available data can be reconciled in the context of higher-order assembly formation, and that TIR domains function through signaling by cooperative assembly formation (SCAF)

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. rings in NLRs) or open-ended higher-order assemblies (e.g. filamentous assemblies by members of the death-fold family and TIR domains). This signaling by cooperative assembly formation (SCAF) mechanism allows rapid and strongly amplified responses to minute amounts of stimulus. While the characterization of the molecular mechanisms of SCAF has seen rapid progress, little is known about its regulation. One emerging theme involves proteins produced both in host cells and by pathogens that appear to mimic the signaling components. Recently characterized examples involve the capping of the filamentous assemblies formed by caspase-1 CARDs by the CARD-only protein INCA, and those formed by caspase-8 by the DED-containing protein MC159. By contrast, the CARD-only protein ICEBERG and the DED-containing protein cFLIP incorporate into signaling filaments and presumably interfere with proximity based activation of caspases. We review selected examples of SCAF in innate immunity pathways and focus on the current knowledge on signaling component mimics produced by mammalian and pathogen cells and what is known about their mechanisms of action

Crystallization and X-ray diffraction analysis of the N-terminal domain of the Toll-like receptor signalling adaptor protein TRIF/TICAM-1

As part of the mammalian innate immune response, Toll-like receptors 3 and 4 can signal via the adaptor protein TRIF/TICAM-1 to elicit the production of type-I interferons and cytokines. Recent studies have suggested an auto-inhibitory role for the N-terminal domain (NTD) of TRIF. This domain has no significant sequence similarity to proteins of known structure. In this paper, the crystallization and X-ray diffraction analysis of TRIF-NTD and its selenomethionine-labelled mutant TRIF-NTDA66M/L113M are reported. Thin plate-like crystals of native TRIF-NTD obtained using polyethylene glycol 3350 as precipitant diffracted X-rays to 1.9 Å resolution. To facilitate phase determination, two additional methionines were incorporated into the protein at positions chosen based on the occurrence of methionines in TRIF homologues in different species. Crystals of the selenomethionine-labelled protein were obtained under conditions similar to the wild-type protein; these crystals diffracted X-rays to 2.5 Å resolution. The TRIF-NTD and TRIF-NTDA66M/L113M crystals have the symmetry of space groups P2 12121 and P1, and most likely contain two and four molecules in the asymmetric unit, respectively. These results provide a sound foundation for the future structure determination of this novel domain

The Methylococcus capsulatus (Bath) Secreted Protein, MopE*, Binds Both Reduced and Oxidized Copper

Under copper limiting growth conditions the methanotrophic bacterium Methylococcus capsulatus (Bath) secrets essentially only one protein, MopE*, to the medium. MopE* is a copper-binding protein whose structure has been determined by X-ray crystallography. The structure of MopE* revealed a unique high affinity copper binding site consisting of two histidine imidazoles and one kynurenine, the latter an oxidation product of Trp130. In this study, we demonstrate that the copper ion coordinated by this strong binding site is in the Cu(I) state when MopE* is isolated from the growth medium of M. capsulatus. The conclusion is based on X-ray Near Edge Absorption spectroscopy (XANES), and Electron Paramagnetic Resonance (EPR) studies. EPR analyses demonstrated that MopE*, in addition to the strong copper-binding site, also binds Cu(II) at two weaker binding sites. Both Cu(II) binding sites have properties typical of non-blue type II Cu (II) centres, and the strongest of the two Cu(II) sites is characterised by a relative high hyperfine coupling of copper (