Structural Analysis of dsRNA Binding to Anti-viral Pattern Recognition Receptors LGP2 and MDA5.

RIG-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.

Porin self-association enables cell-to-cell contact in Providencia stuartii floating communities.

The gram-negative pathogen Providencia stuartii forms floating communities within which adjacent cells are in apparent contact, before depositing as canonical surface-attached biofilms. Because porins are the most abundant proteins in the outer membrane of gram-negative bacteria, we hypothesized that they could be involved in cell-to-cell contact and undertook a structure-function relationship study on the two porins of P. stuartii, Omp-Pst1 and Omp-Pst2. Our crystal structures reveal that these porins can self-associate through their extracellular loops, forming dimers of trimers (DOTs) that could enable cell-to-cell contact within floating communities. Support for this hypothesis was obtained by studying the porin-dependent aggregation of liposomes and model cells. The observation that facing channels are open in the two porin structures suggests that DOTs could not only promote cell-to-cell contact but also contribute to intercellular communication.

Capped RNA primer binding to influenza polymerase and implications for the mechanism of cap-binding inhibitors.

Influenza polymerase uses short capped primers snatched from nascent Pol II transcripts to initiate transcription of viral mRNAs. Here we describe crystal structures of influenza A and B polymerase bound to a capped primer in a configuration consistent with transcription initiation ('priming state') and show by functional assays that conserved residues from both the PB2 midlink and cap-binding domains are important for positioning the capped RNA. In particular, mutation of PB2 Arg264, which interacts with the triphosphate linkage in the cap, significantly and specifically decreases cap-dependent transcription. We also compare the configuration of the midlink and cap-binding domains in the priming state with their very different relative arrangement (called the 'apo' state) in structures where the potent cap-binding inhibitor VX-787, or a close analogue, is bound. In the 'apo' state the inhibitor makes additional interactions to the midlink domain that increases its affinity beyond that to the cap-binding domain alone. The comparison suggests that the mechanism of resistance of certain mutations that allow virus to escape from VX-787, notably PB2 N510T, can only be rationalized if VX-787 has a dual mode of action, direct inhibition of capped RNA binding as well as stabilization of the transcriptionally inactive 'apo' state.

Structure and function of an insect α-carboxylesterase (αEsterase7) associated with insecticide resistance.

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/ß-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.

Assessing the conformational changes of pb5, the receptor-binding protein of phage T5, upon binding to its Escherichia coli receptor FhuA.

Within tailed bacteriophages, interaction of the receptor-binding protein (RBP) with the target cell triggers viral DNA ejection into the host cytoplasm. In the case of phage T5, the RBP pb5 and the receptor FhuA, an outer membrane protein of Escherichia coli, have been identified. Here, we use small angle neutron scattering and electron microscopy to investigate the FhuA-pb5 complex. Specific deuteration of one of the partners allows the complete masking in small angle neutron scattering of the surfactant and unlabeled proteins when the complex is solubilized in the fluorinated surfactant F6-DigluM. Thus, individual structures within a membrane protein complex can be described. The solution structure of FhuA agrees with its crystal structure; that of pb5 shows an elongated shape. Neither displays significant conformational changes upon interaction. The mechanism of signal transduction within phage T5 thus appears different from that of phages binding cell wall saccharides, for which structural information is available.

Small angle neutron scattering for the study of solubilised membrane proteins.

Small angle neutron scattering (SANS) is a powerful technique for investigating association states and conformational changes of biological macromolecules in solution. SANS is of particular interest for the study of the multi-component systems, as membrane protein complexes, for which in vitro characterisation and structure determination are often difficult. This article details the important physical properties of surfactants in view of small angle neutron scattering studies and the interest to deuterate membrane proteins for contrast variation studies. We present strategies for the production of deuterated membrane proteins and methods for quality control. We then review some studies on membrane proteins, and focus on the strategies to overcome the intrinsic difficulty to eliminate homogeneously the detergent or surfactant signal for solubilised membrane proteins, or that of lipids for membrane proteins inserted in liposomes.

Protein surface and core dynamics show concerted hydration-dependent activation.

By specifically labeling leucine/valine methyl groups and lysine side chains "inside" and "outside" dynamics of proteins on the nanosecond timescale are compared using neutron scattering. Surprisingly, both groups display similar dynamics as a function of temperature, and the buried hydrophobic core is sensitive to hydration and undergoes a dynamical transition.

Structure shows that the BIR2 domain of E3 ligase XIAP binds across the RIPK2 kinase dimer interface.

RIPK2 is an essential adaptor for NOD signalling and its kinase domain is a drug target for NOD-related diseases, such as inflammatory bowel disease. However, recent work indicates that the phosphorylation activity of RIPK2 is dispensable for signalling and that inhibitors of both RIPK2 activity and RIPK2 ubiquitination prevent the essential interaction between RIPK2 and the BIR2 domain of XIAP, the key RIPK2 ubiquitin E3 ligase. Moreover, XIAP BIR2 antagonists also block this interaction. To reveal the molecular mechanisms involved, we combined native mass spectrometry, NMR, and cryo-electron microscopy to determine the structure of the RIPK2 kinase BIR2 domain complex and validated the interface with in cellulo assays. The structure shows that BIR2 binds across the RIPK2 kinase antiparallel dimer and provides an explanation for both inhibitory mechanisms. It also highlights why phosphorylation of the kinase activation loop is dispensable for signalling while revealing the structural role of RIPK2-K209 residue in the RIPK2-XIAP BIR2 interaction. Our results clarify the features of the RIPK2 conformation essential for its role as a scaffold protein for ubiquitination.

Molecular mechanism of influenza A NS1-mediated TRIM25 recognition and inhibition.

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.

Evolution of Protein Quaternary Structure in Response to Selective Pressure for Increased Thermostability.

Oligomerization has been suggested to be an important mechanism for increasing or maintaining the thermostability of proteins. Although it is evident that protein-protein contacts can result in substantial stabilization in many extant proteins, evidence for evolutionary selection for oligomerization is largely indirect and little is understood of the early steps in the evolution of oligomers. A laboratory-directed evolution experiment that selected for increased thermostability in the αE7 carboxylesterase from the Australian sheep blowfly, Lucilia cuprina, resulted in a thermostable variant, LcαE7-4a, that displayed increased levels of dimeric and tetrameric quaternary structure. A trade-off between activity and thermostability was made during the evolution of thermostability, with the higher-order oligomeric species displaying the greatest thermostability and lowest catalytic activity. Analysis of monomeric and dimeric LcαE7-4a crystal structures revealed that only one of the oligomerization-inducing mutations was located at a potential protein-protein interface. This work demonstrates that by imposing a selective pressure demanding greater thermostability, mutations can lead to increased oligomerization and stabilization, providing support for the hypothesis that oligomerization is a viable evolutionary strategy for protein stabilization.