SUMMARY RIP1 and RIP3 kinases are central players in TNF-induced programmed necrosis. Here, we report that the RIP homotypic interaction motifs (RHIMs) of RIP1 and RIP3 mediate the assembly of heterodimeric filamentous structures. The fibrils exhibit classical characteristics of β-amyloids, as shown by Thioflavin T (ThT) and Congo red (CR) binding, circular dichroism, infrared spectroscopy, X-ray diffraction, and solid-state NMR. Structured amyloid cores are mapped in RIP1 and RIP3 that are flanked by regions of mobility. The endogenous RIP1/RIP3 complex isolated from necrotic cells binds ThT, is ultrastable, and has a fibrillar core structure, whereas necrosis is partially inhibited by ThT, CR, and another amyloid dye, HBX. Mutations in the RHIMs of RIP1 and RIP3 that are defective in the interaction compromise cluster formation, kinase activation, and programmed necrosis in vivo. The current study provides insight into the structural changes that occur when RIP kinases are triggered to execute different signaling outcomes and expands the realm of amyloids to complex formation and signaling.

NF-κB (nuclear factor kappa B) family transcription factors are master regulators of immune and inflammatory processes in response to both injury and infection. In the latent state, NF-κBs are sequestered in the cytosol by their inhibitor IκB (inhibitor of NF-κB) proteins. Upon stimulations of innate immune receptors such as Toll-like receptors and cytokine receptors such as those in the TNF (tumor necrosis factor) receptor superfamily, a series of membrane proximal events lead to the activation of the IKK (IκB kinase). Phosphorylation of IκBs results in their proteasomal degradation and the release of NF-κB for nuclear translocation and activation of gene transcription. Here, we review the plethora of structural studies in these NF-κB activation pathways, including the TRAF (TNF receptor–associated factor) proteins, IKK, NF-κB, ubiquitin ligases, and deubiquitinating enzymes. Although these structures only provide snapshots of isolated processes, an emerging picture is that these signaling cascades coalesce into large oligomeric signaling complexes, or signalosomes, for signal propagation.

Signal sequences target proteins for secretion from cells or for integration into cell membranes. As nascent proteins emerge from the ribosome, signal sequences are recognized by the signal recognition particle (SRP), which subsequently associates with its receptor (SR). In this complex, the SRP and SR stimulate each other's GTPase activity, and GTP hydrolysis ensures unidirectional targeting of cargo through a translocation pore in the membrane. To define the mechanism of reciprocal activation, we determined the 1.9 A structure of the complex formed between these two GTPases. The two partners form a quasi-two-fold symmetrical heterodimer. Biochemical analysis supports the importance of the extensive interaction surface. Complex formation aligns the two GTP molecules in a symmetrical, composite active site, and the 3'OH groups are essential for association, reciprocal activation and catalysis. This unique circle of twinned interactions is severed twice on hydrolysis, leading to complex dissociation after cargo delivery.

SUMMARY In the Drosophila immune response, bacterial derived diaminopimelic acid-type peptidoglycan binds the receptors PGRP-LC and PGRP-LE, which through interaction with the adaptor protein Imd leads to activation of the NF-κB homolog Relish and robust antimicrobial peptide gene expression. PGRP-LC, PGRP-LE, and Imd each contain a motif with some resemblance to the RIP Homotypic Interaction Motif (RHIM), a domain found in mammalian RIPK proteins forming functional amyloids during necroptosis. Here we found that despite sequence divergence, these Drosophila cryptic RHIMs formed amyloid fibrils in vitro and in cells. Amyloid formation was required for signaling downstream of Imd, and in contrast to the mammalian RHIMs, was not associated with cell death. Furthermore, amyloid formation constituted a regulatable step and could be inhibited by Pirk, an endogenous feedback regulator of this pathway. Thus, diverse sequence motifs are capable of forming amyloidal signaling platforms, and the formation of these platforms may present a regulatory point in multiple biological processes.

Key steps in the export of mRNA from the nucleus to the cytoplasm are the transport through the nuclear pore complex (NPC) and the subsequent remodeling of messenger RNA-protein (mRNP) complexes that occurs at the cytoplasmic side of the NPC. Crucial for these events is the recruitment of the DEAD-box helicase Ddx19 to the cytoplasmic filaments of the NPC that is mediated by the nucleoporin Nup214. Here, we present the crystal structure of the Nup214 N-terminal domain in complex with Ddx19 in its ADPbound state at 2.5 Å resolution. Strikingly, the interaction surfaces are not only evolutionarily conserved but also exhibit strongly opposing surface potentials, with the helicase surface being positively and the Nup214 surface being negatively charged. We speculate that the positively charged surface of the interacting ADP-helicase binds competitively to a segment of mRNA of a linearized mRNP, passing through the NPC on its way to the cytoplasm. As a result, the ADP-helicase would dissociate from Nup214 and replace a single bound protein from the mRNA. One cycle of protein replacement would be accompanied, cooperatively, by nucleotide exchange, ATP hydrolysis, release of the ADP-helicase from mRNA and its rebinding to Nup214. Repeat of these cycles would remove proteins from a mRNP, one at a time, akin to a ratchet mechanism for mRNA export.crystal structure ͉ immunofluorescence localization ͉ mRNA export ͉ mutagenesis ͉ Nup214-helicase complex

The mammalian nuclear pore complex (NPC) is an Ϸ120-MDa proteinaceous assembly consisting of Ϸ30 proteins and is the sole gate in the nuclear envelope. The human protooncogene Nup214 was first identified as a target for chromosomal translocation involved in leukemogenesis. Nup214 is located on the cytoplasmic face of the NPC and is implicated in anchoring the cytoplasmic filaments of the NPC and recruiting the RNA helicase Ddx19. Here, we present the crystal structure of the human Nup214 N-terminal domain at 1.65-Å resolution. The structure reveals a seven-bladed ␤-propeller followed by a 30-residue C-terminal extended peptide segment, which folds back onto the ␤-propeller and binds to its bottom face. The ␤-propeller repeats lack any recognizable sequence motif and are distinguished by extensive insertions between the canonical ␤-strands. We propose a mechanism by which the C-terminal peptide extension is involved in NPC assembly.␤-propeller ͉ mRNA export ͉ leukemia ͉ Dbp5/Ddx19 ͉ nuclear pore complex

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Key steps in mRNA export are the nuclear assembly of messenger ribonucleoprotein particles (mRNPs), the translocation of mRNPs through the nuclear pore complex (NPC), and the mRNP remodeling events at the cytoplasmic side of the NPC. Nup358/RanBP2 is a constituent of the cytoplasmic filaments of the NPC specific to higher eukaryotes and provides a multitude of binding sites for the nucleocytoplasmic transport machinery. Here, we present the crystal structure of the Nup358 N-terminal domain (NTD) at 0.95-Å resolution. The structure reveals an α-helical domain that harbors three central tetratricopeptide repeats (TPR), flanked on each side by an additional solvating amphipathic α helix. Overall, the NTD adopts an unusual extended conformation that lacks the characteristic peptide-binding groove observed in canonical TPR domains. Strikingly, the vast majority of the NTD surface exhibits an evolutionarily conserved, positive electrostatic potential, and we demonstrate that the NTD possesses the capability to bind single-stranded RNA in solution. Together, these data suggest that the NTD contributes to mRNP remodeling events at the cytoplasmic face of the NPC.