Structure of the Ion Channel Kir7.1 and Implications for its Function in Normal and Pathophysiologic States.

Hereditary defects in the function of the Kir7.1 in the retinal pigment epithelium are associated with the ocular diseases retinitis pigmentosa, Leber congenital amaurosis, and snowflake vitreal degeneration. Studies also suggest that Kir7.1 may be regulated by a GPCR, the melanocortin-4 receptor, in certain hypothalamic neurons. We present the first structures of human Kir7.1 and describe the conformational bias displayed by two pathogenic mutations, R162Q and E276A, to provide an explanation for the basis of disease and illuminate the gating pathway. We also demonstrate the structural basis for the blockade of the channel by a small molecule ML418 and demonstrate that channel blockade in vivo activates MC4R neurons in the paraventricular nucleus of the hypothalamus (PVH), inhibiting food intake and inducing weight loss. Preliminary purification, and structural and pharmacological characterization of an in tandem construct of MC4R and Kir7.1 suggests that the fusion protein forms a homotetrameric channel that retains regulation by liganded MC4R molecules.

The unique structural characteristics of the Kir 7.1 inward rectifier potassium channel: a novel player in energy homeostasis control.

The inward rectifier potassium channel Kir7.1, encoded by the KCNJ13 gene, is a tetramer composed of two-transmembrane domain-spanning monomers, closer in homology to Kir channels associated with potassium transport such as Kir1.1, 1.2, and 1.3. Compared with other channels, Kir7.1 exhibits small unitary conductance and low dependence on external potassium. Kir7.1 channels also show a phosphatidylinositol 4,5-bisphosphate (PIP2) dependence for opening. Accordingly, retinopathy-associated Kir7.1 mutations mapped at the binding site for PIP2 resulted in channel gating defects leading to channelopathies such as snowflake vitreoretinal degeneration and Leber congenital amaurosis in blind patients. Lately, this channel's role in energy homeostasis was reported due to the direct interaction with the melanocortin type 4 receptor (MC4R) in the hypothalamus. As this channel seems to play a multipronged role in potassium homeostasis and neuronal excitability, we will discuss what is predicted from a structural viewpoint and its possible implications for hunger control.

Membrane orientation and oligomerization of the melanocortin receptor accessory protein 2.

The melanocortin receptor accessory protein 2 (MRAP2) plays a pivotal role in the regulation of several G protein-coupled receptors that are essential for energy balance and food intake. MRAP2 loss-of-function results in obesity in mammals. MRAP2 and its homolog MRAP1 have an unusual membrane topology and are the only known eukaryotic proteins that thread into the membrane in both orientations. In this study, we demonstrate that the conserved polybasic motif that dictates the membrane topology and dimerization of MRAP1 does not control the membrane orientation and dimerization of MRAP2. We also show that MRAP2 dimerizes through its transmembrane domain and can form higher-order oligomers that arrange MRAP2 monomers in a parallel orientation. Investigating the molecular details of MRAP2 structure is essential for understanding the mechanism by which it regulates G protein-coupled receptors and will aid in elucidating the pathways involved in metabolic dysfunction.

Determination of the melanocortin-4 receptor structure identifies Ca2+ as a cofactor for ligand binding.

The melanocortin-4 receptor (MC4R) is involved in energy homeostasis and is an important drug target for syndromic obesity. We report the structure of the antagonist SHU9119-bound human MC4R at 2.8-angstrom resolution. Ca2+ is identified as a cofactor that is complexed with residues from both the receptor and peptide ligand. Extracellular Ca2+ increases the affinity and potency of the endogenous agonist α-melanocyte-stimulating hormone at the MC4R by 37- and 600-fold, respectively. The ability of the MC4R crystallized construct to couple to ion channel Kir7.1, while lacking cyclic adenosine monophosphate stimulation, highlights a heterotrimeric GTP-binding protein (G protein)-independent mechanism for this signaling modality. MC4R is revealed as a structurally divergent G protein-coupled receptor (GPCR), with more similarity to lipidic GPCRs than to the homologous peptidic GPCRs.

Discovery of processive catalysis by an exo-hydrolase with a pocket-shaped active site.

Substrates associate and products dissociate from enzyme catalytic sites rapidly, which hampers investigations of their trajectories. The high-resolution structure of the native Hordeum exo-hydrolase HvExoI isolated from seedlings reveals that non-covalently trapped glucose forms a stable enzyme-product complex. Here, we report that the alkyl ß-D-glucoside and methyl 6-thio-ß-gentiobioside substrate analogues perfused in crystalline HvExoI bind across the catalytic site after they displace glucose, while methyl 2-thio-ß-sophoroside attaches nearby. Structural analyses and multi-scale molecular modelling of nanoscale reactant movements in HvExoI reveal that upon productive binding of incoming substrates, the glucose product modifies its binding patterns and evokes the formation of a transient lateral cavity, which serves as a conduit for glucose departure to allow for the next catalytic round. This path enables substrate-product assisted processive catalysis through multiple hydrolytic events without HvExoI losing contact with oligo- or polymeric substrates. We anticipate that such enzyme plasticity could be prevalent among exo-hydrolases.

2D Projection Analysis of GPCR Complexes by Negative Stain Electron Microscopy.

While electron cryo-microscopy (cryo-EM) of biological specimens is the preferred single particle EM method for structure determination, its application is very challenging for the typically small (<150 kDa) complexes between GPCRs and their partner proteins. Negative stain EM, whereby the biological samples are embedded in a thin layer of heavy metal solution, is a well-established alternative technique that provides the enhanced contrast needed to visualize small macromolecular complexes. This methodology can offer a simple and powerful tool for the rapid evaluation of sample characteristics, such as homogeneity or oligomeric state. When coupled to single particle classification and averaging, negative stain EM can provide valuable information on the overall architecture and dynamics of protein complexes. Here we provide a concise protocol for negative stain imaging and two-dimensional (2D) projection analysis of GPCR complexes, including notes for the intricacies of the application in these biological systems.

ATP-dependent effector-like functions of RIG-I-like receptors.

The vertebrate antiviral innate immune system is often considered to consist of two distinct groups of proteins: pattern recognition receptors (PRRs) that detect viral infection and induce the interferon (IFN) signaling, and effectors that directly act against viral replication. Accordingly, previous studies on PRRs, such as RIG-I and MDA5, have primarily focused on their functions in viral double-stranded RNA (dsRNA) detection and consequent antiviral signaling. We report here that both RIG-I and MDA5 efficiently displace viral proteins pre-bound to dsRNA in a manner dependent on their ATP hydrolysis, and that this activity assists a dsRNA-dependent antiviral effector protein, PKR, and allows RIG-I to promote MDA5 signaling. Furthermore, truncated RIG-I/MDA5 lacking the signaling domain, and hence the IFN stimulatory activity, displaces viral proteins and suppresses replication of certain viruses in an ATP-dependent manner. Thus, this study reveals novel "effector-like" functions of RIG-I and MDA5 that challenge the conventional view of PRRs.

Molecular imprinting as a signal-activation mechanism of the viral RNA sensor RIG-I.

RIG-I activates interferon signaling pathways by promoting filament formation of the adaptor molecule, MAVS. Assembly of the MAVS filament is mediated by its CARD domain (CARD(MAVS)), and requires its interaction with the tandem CARDs of RIG-I (2CARD(RIG-I)). However, the precise nature of the interaction between 2CARD(RIG-I) and CARD(MAVS), and how this interaction leads to CARD(MAVS) filament assembly, has been unclear. Here we report a 3.6 Å electron microscopy structure of the CARD(MAVS) filament and a 3.4 Å crystal structure of the 2CARD(RIG-I):CARD(MAVS) complex, representing 2CARD(RIG-I) "caught in the act" of nucleating the CARD(MAVS) filament. These structures, together with functional analyses, show that 2CARD(RIG-I) acts as a template for the CARD(MAVS) filament assembly, by forming a helical tetrameric structure and recruiting CARD(MAVS) along its helical trajectory. Our work thus reveals that signal activation by RIG-I occurs by imprinting its helical assembly architecture on MAVS, a previously uncharacterized mechanism of signal transmission.

Structural basis for ubiquitin-mediated antiviral signal activation by RIG-I.

Ubiquitin (Ub) has important roles in a wide range of intracellular signalling pathways. In the conventional view, ubiquitin alters the signalling activity of the target protein through covalent modification, but accumulating evidence points to the emerging role of non-covalent interaction between ubiquitin and the target. In the innate immune signalling pathway of a viral RNA sensor, RIG-I, both covalent and non-covalent interactions with K63-linked ubiquitin chains (K63-Ubn) were shown to occur in its signalling domain, a tandem caspase activation and recruitment domain (hereafter referred to as 2CARD). Non-covalent binding of K63-Ubn to 2CARD induces its tetramer formation, a requirement for downstream signal activation. Here we report the crystal structure of the tetramer of human RIG-I 2CARD bound by three chains of K63-Ub2. 2CARD assembles into a helical tetramer resembling a 'lock-washer', in which the tetrameric surface serves as a signalling platform for recruitment and activation of the downstream signalling molecule, MAVS. Ubiquitin chains are bound along the outer rim of the helical trajectory, bridging adjacent subunits of 2CARD and stabilizing the 2CARD tetramer. The combination of structural and functional analyses reveals that binding avidity dictates the K63-linkage and chain-length specificity of 2CARD, and that covalent ubiquitin conjugation of 2CARD further stabilizes the Ub-2CARD interaction and thus the 2CARD tetramer. Our work provides unique insights into the novel types of ubiquitin-mediated signal-activation mechanism, and previously unexpected synergism between the covalent and non-covalent ubiquitin interaction modes.

RIG-I forms signaling-competent filaments in an ATP-dependent, ubiquitin-independent manner.

Retinoic acid-inducible gene 1 (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) are paralogous receptors for viral double-stranded RNA (dsRNA) with divergent specificity. We have previously shown that MDA5 forms filaments upon viral dsRNA recognition and that this filament formation is essential for interferon signal activation. Here, we show that while RIG-I binds to a dsRNA end as a monomer in the absence of ATP, it assembles in the presence of ATP into a filament that propagates from the dsRNA end to the interior. Furthermore, RIG-I filaments directly stimulate mitochondrial antiviral signaling (MAVS) filament formation without any cofactor, such as polyubiquitin chains, and forced juxtaposition of the isolated signaling domain of RIG-I, as it would be in the filament, is sufficient to activate interferon signaling. Our findings thus define filamentous architecture as a common yet versatile molecular platform for divergent viral RNA detection and proximity-induced signal activation by RIG-I and MDA5.

Structural basis for dsRNA recognition, filament formation, and antiviral signal activation by MDA5.

MDA5, a viral double-stranded RNA (dsRNA) receptor, shares sequence similarity and signaling pathways with RIG-I yet plays essential functions in antiviral immunity through distinct specificity for viral RNA. Revealing the molecular basis for the functional divergence, we report here the crystal structure of MDA5 bound to dsRNA, which shows how, using the same domain architecture, MDA5 recognizes the internal duplex structure, whereas RIG-I recognizes the terminus of dsRNA. We further show that MDA5 uses direct protein-protein contacts to stack along dsRNA in a head-to-tail arrangement, and that the signaling domain (tandem CARD), which decorates the outside of the core MDA5 filament, also has an intrinsic propensity to oligomerize into an elongated structure that activates the signaling adaptor, MAVS. These data support a model in which MDA5 uses long dsRNA as a signaling platform to cooperatively assemble the core filament, which in turn promotes stochastic assembly of the tandem CARD oligomers for signaling.

Multi-level regulation of cellular recognition of viral dsRNA.

Effective antiviral immunity depends on accurate recognition of viral RNAs by the innate immune system. Double-stranded RNA (dsRNA) often accumulates in virally infected cells and was initially considered a unique viral signature that was sufficient to initiate antiviral response through dsRNA receptors and dsRNA-dependent effectors such as Toll-like receptor 3, retinoic acid inducible gene-1, protein kinase RNA-activated and oligoadenylate synthetase. However, dsRNA is also present in many cellular RNAs, raising a question of how these receptors and effectors discriminate between viral and cellular dsRNAs. Accumulating evidence suggests that innate immune sensors detect not only dsRNA structure but also other and often multiple features of RNA such as length, sequence, cellular location, post-transcriptional processing and modification, which are divergent between viral and cellular RNAs. This review summarizes recent findings on the substrate specificities of a few selected dsRNA-dependent effectors and receptors, which have revealed more complex mechanisms involved in cellular discrimination between self and non-self RNA.

MDA5 detects the double-stranded RNA replicative form in picornavirus-infected cells.

RIG-I and MDA5 are cytosolic RNA sensors that play a critical role in innate antiviral responses. Major advances have been made in identifying RIG-I ligands, but our knowledge of the ligands for MDA5 remains restricted to data from transfection experiments mostly using poly(I:C), a synthetic dsRNA mimic. Here, we dissected the IFN-α/ß-stimulatory activity of different viral RNA species produced during picornavirus infection, both by RNA transfection and in infected cells in which specific steps of viral RNA replication were inhibited. Our results show that the incoming genomic plus-strand RNA does not activate MDA5, but minus-strand RNA synthesis and production of the 7.5 kbp replicative form trigger a strong IFN-α/ß response. IFN-α/ß production does not rely on plus-strand RNA synthesis and thus generation of the partially double-stranded replicative intermediate. This study reports MDA5 activation by a natural RNA ligand under physiological conditions.

Kinetic mechanism for viral dsRNA length discrimination by MDA5 filaments.

The viral sensor MDA5 distinguishes between cellular and viral dsRNAs by length-dependent recognition in the range of ~0.5-7 kb. The ability to discriminate dsRNA length at this scale sets MDA5 apart from other dsRNA receptors of the immune system. We have shown previously that MDA5 forms filaments along dsRNA that disassemble upon ATP hydrolysis. Here, we demonstrate that filament formation alone is insufficient to explain its length specificity, because the intrinsic affinity of MDA5 for dsRNA depends only moderately on dsRNA length. Instead, MDA5 uses a combination of end disassembly and slow nucleation kinetics to "discard" short dsRNA rapidly and to suppress rebinding. In contrast, filaments on long dsRNA cycle between partial end disassembly and elongation, bypassing nucleation steps. MDA5 further uses this repetitive cycle of assembly and disassembly processes to repair filament discontinuities, which often are present because of multiple, internal nucleation events, and to generate longer, continuous filaments that more accurately reflect the length of the underlying dsRNA scaffold. Because the length of the continuous filament determines the stability of the MDA5-dsRNA interaction, the mechanism proposed here provides an explanation for how MDA5 uses filament assembly and disassembly dynamics to discriminate between self vs. nonself dsRNA.

Cooperative assembly and dynamic disassembly of MDA5 filaments for viral dsRNA recognition.

MDA5, an RIG-I-like helicase, is a conserved cytoplasmic viral RNA sensor, which recognizes dsRNA from a wide-range of viruses in a length-dependent manner. It has been proposed that MDA5 forms higher-order structures upon viral dsRNA recognition or during antiviral signaling, however the organization and nature of this proposed oligomeric state is unknown. We report here that MDA5 cooperatively assembles into a filamentous oligomer composed of a repeating segmental arrangement of MDA5 dimers along the length of dsRNA. Binding of MDA5 to dsRNA stimulates its ATP hydrolysis activity with little coordination between neighboring molecules within a filament. Individual ATP hydrolysis in turn renders an intrinsic kinetic instability to the MDA5 filament, triggering dissociation of MDA5 from dsRNA at a rate inversely proportional to the filament length. These results suggest a previously unrecognized role of ATP hydrolysis in control of filament assembly and disassembly processes, thereby autoregulating the interaction of MDA5 with dsRNA, and provides a potential basis for dsRNA length-dependent antiviral signaling.

NFAT signaling and the invention of vertebrates.

The calcium/calcineurin-dependent NFATc family is thought to have arisen following the recombination of an ancient precursor with a Rel domain about 500 million years ago, producing a new group of signaling and transcription factors (the NFATc genes) found only in the genomes of vertebrates. Cell biological, genetic and biochemical evidence indicates that the circuitry of this pathway is well suited for intercalation with older pathways. We propose that this recombination enabled Ca(2+) signals to be redirected to a new transcriptional program, which provided part of the groundwork for vertebrate morphogenesis and organogenesis. This notion predicts that calcineurin-NFAT signaling would be essential for much of vertebrate development. We review recent evidence supporting this prediction and propose a systematic approach to explore aspects of vertebrate morphogenesis.

High-level expression of a soluble and functional fibronectin type II domain from MMP-2 in the Escherichia coli cytoplasm for solution NMR studies.

We report a method for the expression in Escherichia coli of the isolated second type II fibronectin domain from MMP-2 (FNII-2). FNII-2 was expressed as a His(6)thioredoxin-tagged fusion protein in the thioredoxin reductase deficient E. coli strain BL21trxB(DE3), thus allowing disulfide-bond formation. When cultured at 37 degrees C, the expressed protein is located exclusively in the soluble fraction of the E. coli lysate. The fusion protein from the soluble fraction was purified and the His(6)thioredoxin-tag was cleaved by thrombin, resulting in a yield of approximately 40 mg/L. The recombinant FNII-2 was demonstrated to be functional by its ability to bind to gelatin-Sepharose, correct folding of the purified protein was confirmed by NMR spectroscopy. This approach may generally be applicable to all FNII domains and is a significant simplification relative to existing techniques involving refolding from inclusion bodies or expression in the eukaryotic host, Pichia pastoris.