Structural basis of Nav1.7 inhibition by an isoform-selective small-molecule antagonist.

Voltage-gated sodium (Nav) channels propagate action potentials in excitable cells. Accordingly, Nav channels are therapeutic targets for many cardiovascular and neurological disorders. Selective inhibitors have been challenging to design because the nine mammalian Nav channel isoforms share high sequence identity and remain recalcitrant to high-resolution structural studies.

Structural Insights into WD-Repeat 48 Activation of Ubiquitin-Specific Protease 46.

Protein ubiquitination patterns are an important component of cellular signaling. The WD-repeat protein WDR48 (USP1-associated factor UAF-1) stimulates activity of ubiquitin-specific proteases USP1, USP12, and USP46. To understand how WDR48 exerts its effect on the USP scaffold, we determined structures of the ternary WDR48:USP46:ubiquitin complex.

Structural analysis of bacterial ABC transporter inhibition by an antibody fragment.

Bacterial ATP-binding cassette (ABC) importers play critical roles in nutrient acquisition and are potential antibacterial targets. However, structural bases for their inhibition are poorly defined. These pathways typically rely on substrate binding proteins (SBPs), which are essential for substrate recognition, delivery, and transporter function.

Structural basis of recognition of interferon-α receptor by tyrosine kinase 2.

Tyrosine kinase 2 (TYK2) is a member of the Janus kinase (JAK) family of nonreceptor tyrosine kinases, which are essential for proper signaling in immune responses and development. Here we present a 2.0-Å-resolution crystal structure of a receptor-binding fragment of human TYK2, encompassing the FERM and SH2 domains, in complex with a so-called 'box2'-containing intracellular peptide motif from the interferon-α receptor chain 1 (IFNAR1).

Dimerization of LTβR by LTα1β2 is necessary and sufficient for signal transduction.

Homotrimeric TNF superfamily ligands signal by inducing trimers of their cognate receptors. As a biologically active heterotrimer, Lymphotoxin(LT)α1β2 is unique in the TNF superfamily. How the three unique potential receptor-binding interfaces in LTα1β2 trigger signaling via LTβ Receptor (LTβR) resulting in lymphoid organogenesis and propagation of inflammatory signals is poorly understood.

Monovalent antibody design and mechanism of action of onartuzumab, a MET antagonist with anti-tumor activity as a therapeutic agent.

Binding of hepatocyte growth factor (HGF) to the receptor tyrosine kinase MET is implicated in the malignant process of multiple cancers, making disruption of this interaction a promising therapeutic strategy. However, targeting MET with bivalent antibodies can mimic HGF agonism via receptor dimerization. To address this limitation, we have developed onartuzumab, an Escherichia coli-derived, humanized, and affinity-matured monovalent monoclonal antibody against MET, generated using the knob-into-hole technology that enables the antibody to engage the receptor in a one-to-one fashion.

The crystal structure of the catalytic domain of the NF-κB inducing kinase reveals a narrow but flexible active site.

The NF-κB inducing kinase (NIK) regulates the non-canonical NF-κB pathway downstream of important clinical targets including BAFF, RANKL, and LTβ. Despite numerous genetic studies associating dysregulation of this pathway with autoimmune diseases and hematological cancers, detailed molecular characterization of this central signaling node has been lacking. We undertook a systematic cloning and expression effort to generate soluble, well-behaved proteins encompassing the kinase domains of human and murine NIK.

Molecular basis of Tank-binding kinase 1 activation by transautophosphorylation.

Tank-binding kinase (TBK)1 plays a central role in innate immunity: it serves as an integrator of multiple signals induced by receptor-mediated pathogen detection and as a modulator of IFN levels. Efforts to better understand the biology of this key immunological factor have intensified recently as growing evidence implicates aberrant TBK1 activity in a variety of autoimmune diseases and cancers. Nevertheless, key molecular details of TBK1 regulation and substrate selection remain unanswered.

Structural basis for the dual recognition of helical cytokines IL-34 and CSF-1 by CSF-1R.

Lacking any discernible sequence similarity, interleukin-34 (IL-34) and colony stimulating factor 1 (CSF-1) signal through a common receptor CSF-1R on cells of mononuclear phagocyte lineage. Here, the crystal structure of dimeric IL-34 reveals a helical cytokine fold homologous to CSF-1, and we further show that the complex architecture of IL-34 bound to the N-terminal immunoglobulin domains of CSF-1R is similar to the CSF-1/CSF-1R assembly. However, unique conformational adaptations in the receptor domain geometry and intermolecular interface explain the cross-reactivity of CSF-1R for two such distantly related ligands.

Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity.

The Ras gene is frequently mutated in cancer, and mutant Ras drives tumorigenesis. Although Ras is a central oncogene, small molecules that bind to Ras in a well-defined manner and exert inhibitory effects have not been uncovered to date. Through an NMR-based fragment screen, we identified a group of small molecules that all bind to a common site on Ras.

Phosphorylation-dependent activity of the deubiquitinase DUBA.

Addition and removal of ubiquitin or ubiquitin chains to and from proteins is a tightly regulated process that contributes to cellular signaling and protein stability. Here we show that phosphorylation of the human deubiquitinase DUBA (OTUD5) at a single residue, Ser177, is both necessary and sufficient to activate the enzyme. The crystal structure of the ubiquitin aldehyde adduct of active DUBA reveals a marked cooperation between phosphorylation and substrate binding.

Multiple novel classes of APRIL-specific receptor-blocking peptides isolated by phage display.

A proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor (TNF) ligand superfamily and has a proliferative effect on both normal and tumor cells. The TNF family receptors (B-cell maturation antigen (BCMA), transmembrane activator and CAML-interactor (TACI), and BAFF receptor-3 (BR3)) for APRIL and the closely related ligand, B-cell activating factor of the TNF family (BAFF), bind these ligands through a highly conserved six residue DXL motif ((F/Y/W)-D-X-L-(V/T)-(R/G)). Panning peptide phage display libraries led to the identification of several novel classes of APRIL-binding peptides, which could be grouped by their common sequence motifs.

Engineering human IgG1 affinity to human neonatal Fc receptor: impact of affinity improvement on pharmacokinetics in primates.

The pH-dependent binding of Igs to the neonatal FcR (FcRn) plays a critical role in the in vivo homeostasis of IgGs. Modulating the interaction between Fc and FcRn through protein engineering is one method for improving the pharmacokinetics of therapeutic Abs. Recent studies disputed the direct relationship between increasing FcRn affinity and improved pharmacokinetic properties.

Anti-BR3 antibodies: a new class of B-cell immunotherapy combining cellular depletion and survival blockade.

Removal of pathogenic B lymphocytes by depletion of monoclonal antibodies (mAbs) or deprivation of B-cell survival factors has demonstrated clinical benefit in both oncologic and immunologic diseases. Partial clinical responses and emerging data demonstrating incomplete B-cell depletion after immunotherapy fuels the need for improved therapeutic modalities. Lessons from the first generation of therapeutics directed against B-cell-specific antigens (CD20, CD22) are being applied to develop novel antibodies with additional functional attributes.

Synthetic anti-BR3 antibodies that mimic BAFF binding and target both human and murine B cells.

BR3, which is expressed on all mature B cells, is a specific receptor for the B-cell survival and maturation factor BAFF (B-cell-activating factor belonging to the tumor necrosis factor [TNF] family). In order to investigate the consequences of targeting BR3 in murine models and to assess the potential of BR3 antibodies as human therapeutics, synthetic antibody phage libraries were employed to identify BAFF-blocking antibodies cross-reactive to murine and human BR3, which share 52% identity in their extracellular domains. We found an antibody, CB1, which exhibits muM affinity for murine BR3 and very weak affinity for the human receptor.

Structures of APRIL-receptor complexes: like BCMA, TACI employs only a single cysteine-rich domain for high affinity ligand binding.

TACI is a member of the tumor necrosis factor receptor superfamily and serves as a key regulator of B cell function. TACI binds two ligands, APRIL and BAFF, with high affinity and contains two cysteine-rich domains (CRDs) in its extracellular region; in contrast, BCMA and BR3, the other known high affinity receptors for APRIL and BAFF, respectively, contain only a single or partial CRD. However, another form of TACI exists wherein the N-terminal CRD is removed by alternative splicing.

The crystal structure of a proliferation-inducing ligand, APRIL.

A proliferation-inducing ligand (APRIL) is a TNF-like cytokine that stimulates tumor cell growth. Within the TNF ligand superfamily, APRIL is most similar to B-cell activation factor (BAFF) with which it shares 30% sequence identity. APRIL binds the receptors B-cell maturation antigen (BCMA) and TACI with high affinity; both of these receptors have also been shown to bind BAFF, although BCMA has significantly higher affinity for APRIL than BAFF.

Convergent recognition of the IgE binding site on the high-affinity IgE receptor.

Two structurally distinct classes of peptides were recently identified by phage display that bind the high-affinity IgE receptor, FcepsilonRI, and block IgE binding and subsequent receptor activation. Both classes adopt highly stable structures in solution, one forming a beta hairpin, with the other forming a helical "zeta" structure. Despite these differences, the two classes bind competitively to the same site on the receptor.

Engineering an APRIL-specific B cell maturation antigen.

B cell maturation antigen (BCMA) is a tumor necrosis factor receptor family member whose physiological role remains unclear. BCMA has been implicated as a receptor for both a proliferation-inducing ligand (APRIL) and B cell-activating factor (BAFF), tumor necrosis factor ligands that bind to multiple tumor necrosis factor receptor and have been reported to play a role in autoimmune disease and cancer. The results presented herein provide a dual perspective analysis of BCMA binding to both APRIL and BAFF.

The crystal structures of EDA-A1 and EDA-A2: splice variants with distinct receptor specificity.

EDA is a tumor necrosis factor family member involved in ectodermal development. Splice variants EDA-A1 and EDA-A2 differ only by the presence of Glu 308 and Val 309 in the expected receptor binding region of EDA-A1 but not EDA-A2. This two amino acid difference functions as a switch controlling receptor specificity.

BAFF/BLyS receptor 3 comprises a minimal TNF receptor-like module that encodes a highly focused ligand-binding site.

BAFF/BLyS, a member of the tumor necrosis family (TNF) superfamily of ligands, is a crucial survival factor for B cells. BAFF binds three receptors, TACI, BCMA, and BR3, with signaling through BR3 being essential for promoting B cell function. Typical TNF receptor (TNFR) family members bind their cognate ligands through interactions with two cysteine-rich domains (CRDs).

BAFF/BLyS receptor 3 binds the B cell survival factor BAFF ligand through a discrete surface loop and promotes processing of NF-kappaB2.

The TNF-like ligand BAFF/BLyS is a potent survival factor for B cells. It binds three receptors: TACI, BCMA, and BR3. We show that BR3 signaling promotes processing of the transcription factor NF-kappaB2/p100 to p52.

Stable "zeta" peptides that act as potent antagonists of the high-affinity IgE receptor.

Recently we described a family of peptides, unrelated in sequence to IgE, that form stable beta-hairpins in solution and inhibit IgE activity in the microM range [Nakamura, G. R., Starovasnik, M.

Identification of a novel homotypic interaction motif required for the phosphorylation of receptor-interacting protein (RIP) by RIP3.

Receptor-interacting protein (RIP), a Ser/Thr kinase component of the tumor necrosis factor (TNF) receptor-1 signaling complex, mediates activation of the nuclear factor kappaB (NF-kappaB) pathway. RIP2 and RIP3 are related kinases that share extensive sequence homology with the kinase domain of RIP. Unlike RIP, which has a C-terminal death domain, and RIP2, which has a C-terminal caspase activation and recruitment domain, RIP3 possesses a unique C terminus.