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 displayed by native IMPs. Many proteins remain difficult to study owing to a lack of suitable detergents. We introduce a class of amphiphiles, each of which is 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 display favorable behavior relative to conventional detergents, as tested on 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.

Linear ubiquitin chains are important regulators of cellular signaling pathways that control innate immunity and inflammation through NF-κB activation and protection against TNFα-induced apoptosis1-5. 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 multi-subunit E3 ligase6. 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” ubiquitin7. Here we report the crystal structure of the catalytic core of HOIP in its apo form and in complex with ubiquitin. The C-terminal portion of HOIP adopts a novel fold that, together with a zinc finger, forms an ubiquitin-binding platform which orients the acceptor ubiquitin and positions its α-amino group for nucleophilic attack on the E3~ubiquitin thioester. The carboxy-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-kB pathway in vivo and explain the determinants of linear ubiquitin chain specificity by LUBAC.

Wiley‐VCH, Weinheim, 2011. 524 pp., hardcover, € 75.00.—ISBN 978‐3527324514

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.

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.Membrane proteins (MPs) play crucial roles in biology, but these proteins are difficult to handle and analyze because of their physical properties. 1 The native conformations of MPs display extensive nonpolar surfaces, which is necessary for residence in a lipid bilayer but leads to denaturation and/or aggregation in an aqueous medium. Detergents, such as dodecyl-β-D-maltoside (DDM), are typically employed to render MPs soluble by coating nonpolar protein surfaces.2 However, not all MPs can be maintained in native-like conformations when solubilized with conventional detergents.3 Moreover, even when a native conformation can be achieved, the MP-detergent complex may manifest unfavorable properties with regard to structural analysis (inability to crystallize and/or too large for NMR). Since our understanding of membrane protein structure and function remains poorly developed relative to soluble proteins, there is a persistent need for new amphiphilic "assistants" that can promote solubilization and manipulation of MPs. 4Several groups have reported creative implementations of the "facial amphiphile" concept for the design of novel amphiphiles that display favorable behavior with selected membrane proteins. 5 McGregor et al., for example, have reported lipopeptides that are intended to match the width of a lipid bilayer and to form a sheath around nonpolar surfaces of MPs.5c Zhang et al. have developed cholate-based amphiphiles in which hydrophilic maltose units project from one side of the rigid and hydrophobic steroidal skeleton. 5d Here we disclose the design of "tandem facial amphiphiles" (TFAs), which contain a pair of maltosefunctionalized deoxycholate units. Unlike previous cholate-based designs, the TFAs are long enough to match bilayer width, 7 and unlike lipopeptides, the TFAs are readily synthesized in large quantities. We show that one TFA forms micelles containing only six molecules, and gellman@chem.wisc.edu; Gether@sund.ku.dk; b.byrne@imperial.ac.uk; stroud@msg.ucsf A set of four TFAs was generated from a deoxycholate-bis-maltoside building block via linkage with a diaminopropane unit ( Figure 1). Molecular mechanics calculations suggest that an extended conformation of the TFA backbone has a length that is comparable to the width of a typical lipid bilayer (~30 Å). 7 These TFAs vary in the appendage on the amide nitrogen atoms. Each amphiphile could be obtained in excellent purity (>98%) and good overall yield (~65%) in five straightforward synthetic steps with two chromatographic purifications.7 Multi-gram quantities are readily available.The TFAs displayed interesting behavior in water. TFA-0 forms a hydrogel at concentrations > 0.4 wt %, and this compound was not studied further. The other three TFAs...

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.

Integral membrane proteins play central roles in controlling the flow of information and molecules across membranes. Our understanding of membrane protein structure and function, however, is seriously limited, mainly due to difficulties in handling and analyzing 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 membrane proteins in their native forms and polar solvent molecules. Here we introduce new tripod amphiphiles displaying favourable behaviours toward several membrane protein systems, leading to enhanced protein solubilization and stabililization compared to both conventional detergents and previously-described tripod amphiphiles.

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.DatabaseThe structures were deposited to the PDB with accession numbers PDB ID: 5IJR, 5IYY, 5JHK, 5J1X, 5JGQ, 5JGI.