IL-17-induced dimerization of IL-17RA drives the formation of the IL-17 signalosome to potentiate signaling.

Signaling through innate immune receptors such as the Toll-like receptor (TLR)/interleukin-1 receptor (IL-1R) superfamily proceeds via the assembly of large membrane-proximal complexes or "signalosomes." Although structurally distinct, the IL-17 receptor family triggers cellular responses that are typical of innate immune receptors. The IL-17RA receptor subunit is shared by several members of the IL-17 family. Using a combination of crystallographic, biophysical, and mutational studies, we show that IL-17A, IL-17F, and IL-17A/F induce IL-17RA dimerization. X-ray analysis of the heteromeric IL-17A complex with the extracellular domains of the IL-17RA and IL-17RC receptors reveals that cytokine-induced IL-17RA dimerization leads to the formation of a 2:2:2 hexameric signaling assembly. Furthermore, we demonstrate that the formation of the IL-17 signalosome potentiates IL-17-induced IL-36γ and CXCL1 mRNA expression in human keratinocytes, compared with a dimerization-defective IL-17RA variant.

Structural basis for selective AMPylation of Rac-subfamily GTPases by Bartonella effector protein 1 (Bep1).

Small GTPases of the Ras-homology (Rho) family are conserved molecular switches that control fundamental cellular activities in eukaryotic cells. As such, they are targeted by numerous bacterial toxins and effector proteins, which have been intensively investigated regarding their biochemical activities and discrete target spectra; however, the molecular mechanism of target selectivity has remained largely elusive. Here we report a bacterial effector protein that selectively targets members of the Rac subfamily in the Rho family of small GTPases but none in the closely related Cdc42 or RhoA subfamilies. This exquisite target selectivity of the FIC domain AMP-transferase Bep1 from Bartonella rochalimae is based on electrostatic interactions with a subfamily-specific pair of residues in the nucleotide-binding G4 motif and the Rho insert helix. Residue substitutions at the identified positions in Cdc42 enable modification by Bep1, while corresponding Cdc42-like substitutions in Rac1 greatly diminish modification. Our study establishes a structural understanding of target selectivity toward Rac-subfamily GTPases and provides a highly selective tool for their functional analysis.

Structural Analysis Reveals that the Cytokine IL-17F Forms a Homodimeric Complex with Receptor IL-17RC to Drive IL-17RA-Independent Signaling.

Interleukin-17A (IL-17A), IL-17F, and IL-17A/F heterodimers are key cytokines of the innate and adaptive immune response. Dysregulation of the IL-17 pathway contributes to immune pathology, and it is therefore important to elucidate the molecular mechanisms that govern IL-17 recognition and signaling. The receptor IL-17RC is thought to act in concert with IL-17RA to transduce IL-17A-, IL-17F-, and IL-17A/F-mediated signals. We report the crystal structure of the extracellular domain of human IL-17RC in complex with IL-17F. In contrast to the expected model, we found that IL-17RC formed a symmetrical 2:1 complex with IL-17F, thus competing with IL-17RA for cytokine binding. Using biophysical techniques, we showed that IL-17A and IL-17A/F also form 2:1 complexes with IL-17RC, suggesting the possibility of IL-17RA-independent IL-17 signaling pathways. The crystal structure of the IL-17RC:IL-17F complex provides a structural basis for IL-17F signaling through IL-17RC, with potential therapeutic applications for respiratory allergy and inflammatory bowel diseases.

The human IL-17A/F heterodimer: a two-faced cytokine with unique receptor recognition properties.

IL-17A and IL-17F are prominent members of the IL-17 family of cytokines that regulates both innate and adaptive immunity. IL-17A has been implicated in chronic inflammatory and autoimmune diseases, and anti-IL-17A antibodies have shown remarkable clinical efficacy in psoriasis and psoriatic arthritis patients. IL-17A and IL-17F are homodimeric cytokines that can also form the IL-17A/F heterodimer whose precise role in health and disease remains elusive. All three cytokines signal through the assembly of a ternary complex with the IL-17RA and IL-17RC receptors. Here we report the X-ray analysis of the human IL-17A/F heterodimer that reveals a two-faced cytokine closely mimicking IL-17A as well as IL-17F. We also present the crystal structure of its complex with the IL-17RA receptor. Unexpectedly in view of the much higher affinity of this receptor toward IL-17A, we find that IL-17RA is bound to the "F-face" of the heterodimer in the crystal. Using site-directed mutagenesis, we then demonstrate that IL-17RA can also bind to the "A-face" of IL-17A/F with similar affinity. Further, we show that IL-17RC does not discriminate between the two faces of the cytokine heterodimer either, thus enabling the formation of two topologically-distinct heterotrimeric complexes with potentially different signaling properties.

A Novel Potent Oral Series of VEGFR2 Inhibitors Abrogate Tumor Growth by Inhibiting Angiogenesis.

This paper describes the identification of 6-(pyrimidin-4-yloxy)-naphthalene-1-carboxamides as a new class of potent and selective human vascular endothelial growth factor receptor 2 (VEGFR2) tyrosine kinase inhibitors. In biochemical and cellular assays, the compounds exhibit single-digit nanomolar potency toward VEGFR2. Compounds of this series show good exposure in rodents when dosed orally. They potently inhibit VEGF-driven angiogenesis in a chamber model and rodent tumor models at daily doses of less than 3 mg/kg by targeting the tumor vasculature as demonstrated by ELISA for TIE-2 in lysates or by immunohistochemical analysis. This novel series of compounds shows a potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role.

Adenylylation of Gyrase and Topo IV by FicT Toxins Disrupts Bacterial DNA Topology.

Toxin-antitoxin (TA) modules are ubiquitous molecular switches controlling bacterial growth via the release of toxins that inhibit cell proliferation. Most of these toxins interfere with protein translation, but a growing variety of other mechanisms hints at a diversity that is not yet fully appreciated. Here, we characterize a group of FIC domain proteins as toxins of the conserved and abundant FicTA family of TA modules, and we reveal that they act by suspending control of cellular DNA topology. We show that FicTs are enzymes that adenylylate DNA gyrase and topoisomerase IV, the essential bacterial type IIA topoisomerases, at their ATP-binding site. This modification inactivates both targets by blocking their ATPase activity, and, consequently, causes reversible growth arrest due to the knotting, catenation, and relaxation of cellular DNA. Our results give insight into the regulation of DNA topology and highlight the remarkable plasticity of FIC domain proteins.

Conserved inhibitory mechanism and competent ATP binding mode for adenylyltransferases with Fic fold.

The ubiquitous FIC domain is evolutionarily conserved from bacteria to human and has been shown to catalyze AMP transfer onto protein side-chain hydroxyl groups. Recently, it was predicted that most catalytically competent Fic proteins are inhibited by the presence of an inhibitory helix αinh that is provided by a cognate anti-toxin (class I), or is part of the N- or C-terminal part of the Fic protein itself (classes II and III). In vitro, inhibition is relieved by mutation of a conserved glutamate of αinh to glycine. For the class III bacterial Fic protein NmFic from Neisseria meningitidis, the inhibitory mechanism has been elucidated. Here, we extend above study by including bacterial class I and II Fic proteins VbhT from Bartonella schoenbuchensis and SoFic from Shewanella oneidensis, respectively, and the respective E->G mutants. Comparative enzymatic and crystallographic analyses show that, in all three classes, the ATP substrate binds to the wild-type FIC domains, but with the α-phosphate in disparate and non-competent orientations. In the E->G mutants, however, the tri-phosphate moiety is found reorganized to the same tightly bound structure through a unique set of hydrogen bonds with Fic signature motif residues. The γ-phosphate adopts the location that is taken by the inhibitory glutamate in wild-type resulting in an α-phosphate orientation that can be attacked in-line by a target side-chain hydroxyl group. The latter is properly registered to the Fic active center by main-chain ß-interactions with the ß-hairpin flap. These data indicate that the active site motif and the exposed edge of the flap are both required to form an adenylylation-competent Fic protein.

Adenylylation control by intra- or intermolecular active-site obstruction in Fic proteins.

Fic proteins that are defined by the ubiquitous FIC (filamentation induced by cyclic AMP) domain are known to catalyse adenylylation (also called AMPylation); that is, the transfer of AMP onto a target protein. In mammalian cells, adenylylation of small GTPases through Fic proteins injected by pathogenic bacteria can cause collapse of the actin cytoskeleton and cell death. It is unknown how this potentially deleterious adenylylation activity is regulated in the widespread Fic proteins that are found in all domains of life and that are thought to have critical roles in intrinsic signalling processes. Here we show that FIC-domain-mediated adenylylation is controlled by a conserved mechanism of ATP-binding-site obstruction that involves an inhibitory α-helix (α(inh)) with a conserved (S/T)XXXE(G/N) motif, and that in this mechanism the invariable glutamate competes with ATP γ-phosphate binding. Consistent with this, FIC-domain-mediated growth arrest of bacteria by the VbhT toxin of Bartonella schoenbuchensis is intermolecularly repressed by the VbhA antitoxin through tight binding of its α(inh) to the FIC domain of VbhT, as shown by structure and function analysis. Furthermore, structural comparisons with other bacterial Fic proteins, such as Fic of Neisseria meningitidis and of Shewanella oneidensis, show that α(inh) frequently constitutes an amino-terminal or carboxy-terminal extension to the FIC domain, respectively, partially obstructing the ATP binding site in an intramolecular manner. After mutation of the inhibitory motif in various Fic proteins, including the human homologue FICD (also known as HYPE), adenylylation activity is considerably boosted, consistent with the anticipated relief of inhibition. Structural homology modelling of all annotated Fic proteins indicates that inhibition by α(inh) is universal and conserved through evolution, as the inhibitory motif is present in ∼90% of all putatively adenylylation-active FIC domains, including examples from all domains of life and from viruses. Future studies should reveal how intrinsic or extrinsic factors modulate adenylylation activity by weakening the interaction of α(inh) with the FIC active site.

Fic domain-catalyzed adenylylation: insight provided by the structural analysis of the type IV secretion system effector BepA.

Numerous bacterial pathogens subvert cellular functions of eukaryotic host cells by the injection of effector proteins via dedicated secretion systems. The type IV secretion system (T4SS) effector protein BepA from Bartonella henselae is composed of an N-terminal Fic domain and a C-terminal Bartonella intracellular delivery domain, the latter being responsible for T4SS-mediated translocation into host cells. A proteolysis resistant fragment (residues 10-302) that includes the Fic domain shows autoadenylylation activity and adenylyl transfer onto Hela cell extract proteins as demonstrated by autoradiography on incubation with α-[(32)P]-ATP. Its crystal structure, determined to 2.9-Å resolution by the SeMet-SAD method, exhibits the canonical Fic fold including the HPFxxGNGRxxR signature motif with several elaborations in loop regions and an additional ß-rich domain at the C-terminus. On crystal soaking with ATP/Mg(2+), additional electron density indicated the presence of a PP(i) /Mg(2+) moiety, the side product of the adenylylation reaction, in the anion binding nest of the signature motif. On the basis of this information and that of the recent structure of IbpA(Fic2) in complex with the eukaryotic target protein Cdc42, we present a detailed model for the ternary complex of Fic with the two substrates, ATP/Mg(2+) and target tyrosine. The model is consistent with an in-line nucleophilic attack of the deprotonated side-chain hydroxyl group onto the α-phosphorus of the nucleotide to accomplish AMP transfer. Furthermore, a general, sequence-independent mechanism of target positioning through antiparallel ß-strand interactions between enzyme and target is suggested.

Crystal Structures of Human MdmX (HdmX) in Complex with p53 Peptide Analogues Reveal Surprising Conformational Changes.

p53 tumor suppressor activity is negatively regulated through binding to the oncogenic proteins Hdm2 and HdmX. The p53 residues Leu(26), Trp(23), and Phe(19) are crucial to mediate these interactions. Inhibiting p53 binding to both Hdm2 and HdmX should be a promising clinical approach to reactivate p53 in the cancer setting, but previous studies have suggested that the discovery of dual Hdm2/HdmX inhibitors will be difficult. We have determined the crystal structures at 1.3 A of the N-terminal domain of HdmX bound to two p53 peptidomimetics without and with a 6-chlorine substituent on the indole (which binds in the same subpocket as Trp(23) of p53). The latter compound is the most potent peptide-based antagonist of the p53-Hdm2 interaction yet to be described. The x-ray structures revealed surprising conformational changes of the binding cleft of HdmX, including an "open conformation" of Tyr(99) and unexpected "cross-talk" between the Trp and Leu pockets. Notably, the 6-chloro p53 peptidomimetic bound with high affinity to both HdmX and Hdm2 (K(d) values of 36 and 7 nm, respectively). Our results suggest that the development of potent dual inhibitors for HdmX and Hdm2 should be feasible. They also reveal possible conformational states of HdmX, which should lead to a better prediction of its interactions with potential biological partners.