Structural basis for ligase-specific conjugation of linear ubiquitin chains by HOIP.

Linear ubiquitin chains are important regulators of cellular signalling pathways that control innate immunity and inflammation through nuclear factor (NF)-κB activation and protection against tumour necrosis factor-α-induced apoptosis. They are synthesized by HOIP, which belongs to the RBR (RING-between-RING) family of E3 ligases and is the catalytic component of LUBAC (linear ubiquitin chain assembly complex), a multisubunit E3 ligase. RBR family members act as RING/HECT hybrids, employing RING1 to recognize ubiquitin-loaded E2 while a conserved cysteine in RING2 subsequently forms a thioester intermediate with the transferred or 'donor' ubiquitin. Here we report the crystal structure of the catalytic core of HOIP in its apo form and in complex with ubiquitin. The carboxy-terminal portion of HOIP adopts a novel fold that, together with a zinc-finger, forms a ubiquitin-binding platform that orients the acceptor ubiquitin and positions its α-amino group for nucleophilic attack on the E3∼ubiquitin thioester. The C-terminal tail of a second ubiquitin molecule is located in close proximity to the catalytic cysteine, providing a unique snapshot of the ubiquitin transfer complex containing both donor and acceptor ubiquitin. These interactions are required for activation of the NF-κB pathway in vivo, and they explain the determinants of linear ubiquitin chain specificity by LUBAC.

Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins.

The understanding of integral membrane protein (IMP) structure and function is hampered by the difficulty of handling these proteins. Aqueous solubilization, necessary for many types of biophysical analysis, generally requires a detergent to shield the large lipophilic surfaces of native IMPs. Many proteins remain difficult to study owing to a lack of suitable detergents. We introduce a class of amphiphiles, each built around a central quaternary carbon atom derived from neopentyl glycol, with hydrophilic groups derived from maltose. Representatives of this maltose-neopentyl glycol (MNG) amphiphile family show favorable behavior relative to conventional detergents, as manifested in multiple membrane protein systems, leading to enhanced structural stability and successful crystallization. MNG amphiphiles are promising tools for membrane protein science because of the ease with which they may be prepared and the facility with which their structures may be varied.

Novel tripod amphiphiles for membrane protein analysis.

Integral membrane proteins play central roles in controlling the flow of information and molecules across membranes. Our understanding of membrane protein structures and functions, however, is seriously limited, mainly due to difficulties in handling and analysing these proteins in aqueous solution. The use of a detergent or other amphipathic agents is required to overcome the intrinsic incompatibility between the large lipophilic surfaces displayed by the membrane proteins in their native forms and the polar solvent molecules. Here, we introduce new tripod amphiphiles displaying favourable behaviours toward several membrane protein systems, leading to an enhanced protein solubilisation and stabilisation compared to both conventional detergents and previously described tripod amphiphiles.

Bacterial TIR-containing proteins and host innate immune system evasion.

The innate immune system provides the first line of host defence against invading pathogens. Key to upregulation of the innate immune response are Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns (PAMPs) and trigger a signaling pathway culminating in the production of inflammatory mediators. Central to this TLR signaling pathway are heterotypic protein-protein interactions mediated through Toll/interleukin-1 receptor (TIR) domains found in both the cytoplasmic regions of TLRs and adaptor proteins. Pathogenic bacteria have developed a range of ingenuous strategies to evade the host immune mechanisms. Recent work has identified a potentially novel evasion mechanism involving bacterial TIR domain proteins. Such domains have been identified in a wide range of pathogenic bacteria, and there is evidence to suggest that they interfere directly with the TLR signaling pathway and thus inhibit the activation of NF-κB. The individual TIR domains from the pathogenic bacteria Salmonella enterica serovar Enteritidis, Brucella sp, uropathogenic E. coli and Yersinia pestis have been analyzed in detail. The individual bacterial TIR domains from these pathogenic bacteria seem to differ in their modes of action and their roles in virulence. Here, we review the current state of knowledge on the possible roles and mechanisms of action of the bacterial TIR domains.

Pharmacodynamic synergism contributes to the antiseizure action of cannabidiol and clobazam.

The management of refractory epilepsy involves treatment with more than one antiseizure medication (ASM). Combination of ASMs with distinct mechanisms of action are hypothesized to improve overall treatment effectiveness. In clinical trials, concomitant use of cannabidiol (CBD) and clobazam (CLB) was associated with increased seizure reduction and bidirectional elevation in levels of their active metabolites, 7-hydroxy-cannabidiol (7-OH-CBD) and nor-clobazam (n-CLB). Using isobolographic analysis, we investigated whether CBD and CLB interacted pharmacodynamically. In the mouse maximal electroshock seizure (MES) test, brain tissue levels of CBD and CLB corresponding to seizure prevention in 50% of animals (brain Effective Exposure, bEE50) were 7.9 µM and 1.6 µM, respectively. In the 6 Hz psychomotor seizure model, 7-OH-CBD displayed a 5-fold greater potency than CBD (b-EE50, 8.7 µM vs 47.3 µM). Isobolographic analysis performed on combination of CBD/CLB at 1:1, 3:1, and 1:3 ratios based on equi-effective bEE50 values revealed synergism at all doses with combination indices (CI) of 0.43, 0.62 and 0.75 respectively. These outcomes were independent of pharmacokinetic interaction between CBD and CLB. These findings identify pharmacodynamic synergism as an important factor underlying enhanced antiseizure effect during concomitant CBD and CLB use.

A Toll/interleukin (IL)-1 receptor domain protein from Yersinia pestis interacts with mammalian IL-1/Toll-like receptor pathways but does not play a central role in the virulence of Y. pestis in a mouse model of bubonic plague.

The Toll/interleukin (IL)-1 receptor (TIR) domain is an essential component of eukaryotic innate immune signalling pathways. Interaction between TIR domains present in Toll-like receptors and associated adaptors initiates and propagates an immune signalling cascade. Proteins containing TIR domains have also been discovered in bacteria. Studies have subsequently shown that these proteins are able to modulate mammalian immune signalling pathways dependent on TIR interactions and that this may represent an evasion strategy for bacterial pathogens. Here, we investigate a TIR domain protein from the highly virulent bacterium Yersinia pestis, the causative agent of plague. When overexpressed in vitro this protein is able to downregulate IL-1ß- and LPS-dependent signalling to NFκB and to interact with the TIR adaptor protein MyD88. This interaction is dependent on a single proline residue. However, a Y. pestis knockout mutant lacking the TIR domain protein was not attenuated in virulence in a mouse model of bubonic plague. Minor alterations in the host cytokine response to the mutant were indicated, suggesting a potential subtle role in pathogenesis. The Y. pestis mutant also showed increased auto-aggregation and reduced survival in high-salinity conditions, phenotypes which may contribute to pathogenesis or survival.

Yersinia pestis TIR-domain protein forms dimers that interact with the human adaptor protein MyD88.

Recent research has highlighted the presence of Toll/Interleukin 1 receptor (TIR)-domain proteins (Tdps) in a range of bacteria, suggested to form interactions with the human adaptor protein MyD88 and inhibit intracellular signaling from Toll-like receptors (TLRs). A Tdp has been identified in Yersinia pestis (YpTdp), a highly pathogenic bacterium responsible for plague. Expression of a number of YpTIR constructs of differing lengths (YpTIR1, S130-A285; YpTIR2, I137-I273; YpTIR3, I137-246; YpTIR4, D107-S281) as fusions with an N-terminal GB1 tag (the B1 immunoglobulin domain of Streptococcal protein G) yielded high levels of soluble protein. Subsequent purification yielded 4-6 mg/L pure, folded protein. Thrombin cleavage allowed separation of the GB1 tag from YpTIR4 resulting in folded protein after cleavage. Nuclear magnetic resonance spectroscopy, size exclusion chromatography, SDS-PAGE analysis and static light scattering all indicate that the YpTIR forms dimers. Generation of a double Cys-less mutant resulted in an unstable protein containing mainly monomers indicating the importance of disulphide bonds in dimer formation. In addition, the YpTIR constructs have been shown to interact with the human adaptor protein MyD88 using 2D NMR and GST pull down. YpTIR is an excellent candidate for further study of the mechanism of action of pathogenic bacterial Tdps.

Tandem facial amphiphiles for membrane protein stabilization.

We describe a new type of synthetic amphiphile that is intended to support biochemical characterization of intrinsic membrane proteins. Members of this new family displayed favorable behavior with four of five membrane proteins tested, and these amphiphiles formed relatively small micelles.

Architecture and hydration of the arginine-binding site of neuropilin-1.

Neuropilin-1 (NRP1) is a transmembrane co-receptor involved in binding interactions with variety of ligands and receptors, including receptor tyrosine kinases. Expression of NRP1 in several cancers correlates with cancer stages and poor prognosis. Thus, NRP1 has been considered a therapeutic target and is the focus of multiple drug discovery initiatives. Vascular endothelial growth factor (VEGF) binds to the b1 domain of NRP1 through interactions between the C-terminal arginine of VEGF and residues in the NRP1-binding site including Tyr297, Tyr353, Asp320, Ser346 and Thr349. We obtained several complexes of the synthetic ligands and the NRP1-b1 domain and used X-ray crystallography and computational methods to analyse atomic details and hydration profile of this binding site. We observed side chain flexibility for Tyr297 and Asp320 in the six new high-resolution crystal structures of arginine analogues bound to NRP1. In addition, we identified conserved water molecules in binding site regions which can be targeted for drug design. The computational prediction of the VEGF ligand-binding site hydration map of NRP1 was in agreement with the experimentally derived, conserved hydration structure. Displacement of certain conserved water molecules by a ligand's functional groups may contribute to binding affinity, whilst other water molecules perform as protein-ligand bridges. Our report provides a comprehensive description of the binding site for the peptidic ligands' C-terminal arginines in the b1 domain of NRP1, highlights the importance of conserved structural waters in drug design and validates the utility of the computational hydration map prediction method in the context of neuropilin. DATABASE: The structures were deposited to the PDB with accession numbers PDB ID: 5IJR, 5IYY, 5JHK, 5J1X, 5JGQ, 5JGI.

Glucose-neopentyl glycol (GNG) amphiphiles for membrane protein study.

The development of a new class of surfactants for membrane protein manipulation, "GNG amphiphiles", is reported. These amphiphiles display promising behavior for membrane proteins, as demonstrated recently by the high resolution structure of a sodium-pumping pyrophosphatase reported by Kellosalo et al. (Science, 2012, 337, 473).