RIG-I is a viral RNA sensor that induces the production of type I interferon (IFN) in response to infection with a variety of viruses. Modification of RIG-I with K63-linked poly-ubiquitin chains, synthesised by TRIM25, is crucial for activation of the RIG-I/MAVS signalling pathway. TRIM25 activity is targeted by influenza A virus non-structural protein 1 (NS1) to suppress IFN production and prevent an efficient host immune response. Here we present structures of the human TRIM25 coiled-coil-PRYSPRY module and of complexes between the TRIM25 coiled-coil domain and NS1. These structures show that binding of NS1 interferes with the correct positioning of the PRYSPRY domain of TRIM25 required for substrate ubiquitination and provide a mechanistic explanation for how NS1 suppresses RIG-I ubiquitination and hence downstream signalling. In contrast, the formation of unanchored K63-linked poly-ubiquitin chains is unchanged by NS1 binding, indicating that RING dimerisation of TRIM25 is not affected by NS1.

SummaryRIG-I and MDA5 sense virus-derived short 5′ppp blunt-ended or long dsRNA, respectively, causing interferon production. Non-signaling LGP2 appears to positively and negatively regulate MDA5 and RIG-I signaling, respectively. Co-crystal structures of chicken (ch) LGP2 with dsRNA display a fully or semi-closed conformation depending on the presence or absence of nucleotide. LGP2 caps blunt, 3′ or 5′ overhang dsRNA ends with 1 bp longer overall footprint than RIG-I. Structures of 1:1 and 2:1 complexes of chMDA5 with short dsRNA reveal head-to-head packing rather than the polar head-to-tail orientation described for long filaments. chLGP2 and chMDA5 make filaments with a similar axial repeat, although less co-operatively for chLGP2. Overall, LGP2 resembles a chimera combining a MDA5-like helicase domain and RIG-I like CTD supporting both stem and end binding. Functionally, RNA binding is required for LGP2-mediated enhancement of MDA5 activation. We propose that LGP2 end-binding may promote nucleation of MDA5 oligomerization on dsRNA.

Insect carboxylesterases from the αEsterase gene cluster, such as αE7 (also known as E3) from the Australian sheep blowfly Lucilia cuprina (LcαE7), play an important physiological role in lipid metabolism and are implicated in the detoxification of organophosphate (OP) insecticides. Despite the importance of OPs to agriculture and the spread of insect-borne diseases, the molecular basis for the ability of α-carboxylesterases to confer OP resistance to insects is poorly understood. In this work, we used laboratory evolution to increase the thermal stability of LcαE7, allowing its overexpression in Escherichia coli and structure determination. The crystal structure reveals a canonical α/β-hydrolase fold that is very similar to the primary target of OPs (acetylcholinesterase) and a unique N-terminal α-helix that serves as a membrane anchor. Soaking of LcαE7 crystals in OPs led to the capture of a crystallographic snapshot of LcαE7 in its phosphorylated state, which allowed comparison with acetylcholinesterase and rationalization of its ability to protect insects against the effects of OPs. Finally, inspection of the active site of LcαE7 reveals an asymmetric and hydrophobic substrate binding cavity that is well-suited to fatty acid methyl esters, which are hydrolyzed by the enzyme with specificity constants (∼10 6 M −1 s −1 ) indicative of a natural substrate.protein engineering | directed evolution | ali-esterase T he demand for greater productivity from agriculture and the avoidance of insect-borne diseases has made efficient control of insect pests increasingly important; in 2007, the world market for insecticides was estimated to be in the order of $11 billion US (1). However, the effectiveness of insecticides is decreasing, with over 500 documented instances of insecticide resistance (2). The development of resistance to organophosphate (OP) (Fig. 1A) pesticides through the action of α-carboxylesterases (α-CBEs) has been documented in many species (3), including the Australian sheep blowfly Lucilia cuprina (4). OPs inhibit acetylcholinesterase (AChE) at cholinergic synapses in the central and peripheral nervous systems, which leads to interminable nerve signal transduction and death (5). However, α-CBEs, such as αE7 in L. cuprina (LcαE7), confer a significant protective effect on insects owing to their ability to bind and slowly hydrolyze OPs (6). It has also been shown that this CBE-mediated OP resistance can be increased through G137D and W251L point mutations (4, 7).Insect α-CBEs, including LcαE7, are thought to play important physiological roles in lipid and xenobiotic metabolism (6,8). They are one of the most abundant protein families in insects, with between 20 and 40 active CBEs predicted to be expressed by the fruit fly Drosphila melanogaster (9, 10). Although the physiological substrates of insect α-CBEs are unknown, recent MS results have shown that D. melanogaster αE7 is expressed in the fat body lipid droplet proteome (11), implying a role in lipid or cholesterol metabolism. This work has been ext...